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首页 > 1,25二羟维生素D3对人外周血B淋巴细胞免疫调节作用的研究.pdf.doc

1,25二羟维生素D3对人外周血B淋巴细胞免疫调节作用的研究.pdf.doc

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1,25二羟维生素D3对人外周血B淋巴细胞免疫调节作用的研究.pdf.doc1,25二羟维生素D3对人外周血B淋巴细胞免疫调节作用的研究.pdf.doc 上海交通大学 博士学位论文 1,25二羟维生素D3对人外周血B淋巴细胞免疫调节作用的研究 姓名:陈盛 申请学位级别:博士 专业:内科学风湿病 指导教师:顾越英;陈顺乐 20070501 上海交通大学 2007届博士论文 符号说明 英文全称 中文全称 k 系统性红斑狼疮 SLE Systemic lupus erythematosus 1,25-二羟维生素 1,25(OH)2D3 1,25-Dihydroxyvitamin ...
1,25二羟维生素D3对人外周血B淋巴细胞免疫调节作用的研究.pdf.doc
1,25二羟维生素D3对人外周血B淋巴细胞免疫调节作用的研究.pdf.doc 上海交通大学 博士学位 1,25二羟维生素D3对人外周血B淋巴细胞免疫调节作用的研究 姓名:陈盛 申请学位级别:博士 专业:内科学风湿病 指导教师:顾越英;陈顺乐 20070501 上海交通大学 2007届博士论文 符号说明 英文全称 中文全称 k 系统性红斑狼疮 SLE Systemic lupus erythematosus 1,25-二羟维生素 1,25(OH)2D3 1,25-Dihydroxyvitamin D3 D3 25(OH)D3 25-hydroxyvitamin D3 25羟维生素 D3 VDR Vitamin D3 receptor 维生素 D受体 抗磷脂抗体 CDK Antiphospholipid antibody 25(OH)D3-1-α-hydroxylase CYP27B1 25羟维生素 D3-1-α-羟化酶 ELISA Enzyme-linked Immunosorbent Assay 酶联免疫吸附检测 CYP24A1 24-hydroxylase 24-羟化酶 DC 树突状细胞 Dentritic cell 抗核抗体 ANA Anti-nuclear Antibodies 类风湿关节炎 RA Rheumatoid Arthritis (RA) SLEDAI Systemic Lupus Erythematosus 系统性红斑狼疮疾病活动 指数 Disease activity Index 单克隆抗体 mAb Monoclonal antibody 放射免疫测定法 RIA radioimmunoassay assays CFSE Carboxy-fluorescein diacetate, 羧基荧光素二醋酸盐琥珀 succinimidyl ester 酰亚胺酯 荧光激活细胞分选器 FACS Fluorescence Activated Cell Sorter 固相酶联免疫斑点技术 ELISPOT Enzyme-Linked ImmunoSpot Assay 类转变重组 CRS Class-switch recombination 6 上海交通大学 学位论文原创性声明 本人郑重声明:所呈交的学位论文,是本人在导师的指导下,独立进行研究工作所取得的成果。 除文中已经注明引用的内容外,本论文不包含任何其他个人或集体已经发表或撰写过的作品成 果。对本文的研究做出重要贡献的个人和集体,均已在文中以明确方式标明。本人完全意识到 本声明的法律结果由本人承担。 学位论文作者签名: 日期: 年 月 日 I 上海交通大学 2007届博士论文 上海交通大学 学位论文版权使用授权书 本学位论文作者完全了解学校有关保留、使用学位论文的规定,同意学校保留并向国家有关部 门或机构送交论文的复印件和电子版,允许论文被查阅和借阅。本人授权上海交通大学可以将 本学位论文的全部或部分内容编入有关数据库进行检索,可以采用影印、缩印或扫描等复制手 段保存和汇编本学位论文。 保密?,在 年解密后适用本授权书。 本学位论文属于 不保密?。 (请在以上方框内打“?”) 学位论文作者签名: 指导教师签名: I 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,, , , , , , , , , , , 1,25二羟维生素 D3对人外周血 B淋巴细胞 免疫调节作用的研究 摘要 1,25-二羟维生素 D3(1,25(OH)2D3)能通过抑制树突状细胞(DC)和 T淋巴细胞的生长和分化对免疫功能进行调节。然而,1,25(OH)2D3是否对B 细胞也有直接调节作用,至今尚不明确。 首先,我们检测了系统性红斑狼疮患者 (SLE)血浆1,25(OH)2D3水平 并探讨其临床相关性,发现SLE患者的1,25(OH)2D3水平明显低于健康人群, 并且与疾病活动度、抗核抗体的产生呈明显的负相关,由此提示维生素D可 能在B细胞分化和抗体产生中起到一定作用。 为进一步验证这一观点,我们探索了1,25(OH)2D3对健康人外周血B淋 巴细胞的作用。结果发现1,25(OH)2D3能抑制B细胞增生并诱导其凋亡,能 抑制B细胞分化形成类转变重组后(class-switch recombination)的记忆B细 胞和浆细胞以及减少免疫球蛋白的产生;同时,B细胞自身能表达1α-羟化 酶,后者是1,25(OH)2D3从其前体25(OH)D3完成最后活化所需的关键酶, 由此25(OH)D3同样能对B细胞的功能产生抑制作用。更为重要的是,参与 调节1,25(OH)2D3功能的蛋白包括维生素D受体(VDR)和24-羟化酶的表达, 在1,25(OH)2D3和/或刺激积剂影响下都有不同程度地上调。其后我们发现, 1,25(OH)2D3还能上调细胞周期素依赖性激酶(Cdk)抑制蛋白P27 (而非P18 1 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,, , , , , , , , , , , 和P21)的基因表达,该蛋白可能对调节激活的B细胞增殖及其下游的分化极 为重要。 由此,我们的结果充分证实了1,25(OH)2D3在维持B细胞稳态中起到了 非常重要的作用,而纠正维生素D缺乏可能对治疗B细胞介导的自身免疫性 疾病至关重要。 关键词:1, 25二羟维生素 D3, B细胞,免疫调节,系统性红斑狼疮 2 MODULATORY EFFECTS OF 1,25-DIHYDROXYVITAMIN D3 ON HUMAN PERIPHERAL B CELL ABSTRACT 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) can modulate immune responses by inhibiting growth and differentiation of dendritic cells and T cells. It remains unknown whether 1,25(OH)2D3 can also directly modulate human B cell function. We observed that patients with systemic lupus erythematosus (SLE) have decreased levels of 1,25(OH)2D3 and that the levels were lowest in those with anti-nuclear antibodies (ANA) and increased disease activity suggesting that Vitamin D might play a role in regulating B cell differentiation and antoantibody production. To address this possibility, we examined the effects of 1,25-dihydroxyvitamin D3 on B cell responses. 1,25(OH)2D3 was found to 3 上海交通大学 2007届博士论文 inhibit ongoing proliferation of activated B cells and induce their apoptosis, whereas initial cell division was unimpeded. B cell differentiation, including the generation of immunoglobulin secreting plasma cells and post-switch memory B cells was also significantly inhibited by 1,25(OH)2D3, although upregulation of genetic programs involved in B cell differentiation was only modestly affected. We also noted that human B cells expressed 1α-hydroxylase, the enzyme responsible for converting the precursor 25(OH)D3 to 1,25(OH)2D3 and as a result, B cell responses could be inhibited by the precursor of active Vitamin D. Moreover, proteins involved in regulating the function of 1,25(OH)2D3, namely the Vitamin D receptor and 24-hydroxylase, were upregulated by 1,25(OH)2D3 and/or activation stimuli. Finally, 1,25(OH)2D3 upregulated expression of a number of genes, including p27, but not p18 and p21, that may be important in regulating proliferation of activated B cells and their subsequent differentiation. Our data indicate that 1,25-dihydroxyvitamin D3 may play an important role in the maintenance B cell homeostasis, and that correction of Vitamin D deficiency may be useful in the treatment of B cell mediated autoimmune disorders. KEY WORDS: 1,25-dihydroxyvitamin D3, B cell, Immune modulation, Systemic lupus erythematosus 4 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , 前言 1,25-二羟维生素D3 (1,25(OH)2D3)是维生素D3的生物活性成分,自1923年发现 以来一直用于佝偻病的治疗,在维持骨骼和钙磷代谢中起重要作用。近年来的研究 显示1,25(OH)2D3还具有广泛的免疫调节作用 (1)。虽然维生素D3可以从食物中获得, 但主要的来源是通过皮肤中的光合作用。在紫外线 (270–300 nm)照射下,皮肤中的7 一脱氢胆固醇转化成维生素 D3进入血液,在肝内D3-25-羟化酶的作用下转化成 25- 羟维生素D3 (25(OH)D3)。进一步的羟化则在肾脏近曲小管中完成,在 25(OH)D3-1- 羟化酶 (CYP27B1)作用下生成具有生物活性的 1,25(OH)2D3。24-羟化酶 (CYP24A1) 是其主要的降解酶,可以在 1,25(OH)2D3诱导下产生 (2)。在某些情况下,肾外其他 部位的组织细胞,如活化的巨噬细胞和树突状细胞也能表达 CYP27B1,但这种转变 较肝肾通路其量甚微,目前对这种转变的生理意义尚不清楚,有可能起自分泌或旁 分泌的作用(3, 4)。已知的1,25 (OH)2D3生物学效应主要是通过维生素 D受体(VDR) 介导,现已证明VDR作为核激素受体超家族的一员,不仅存在于经典的靶组织 (如小 肠、骨、肾脏、甲状旁腺 ),各种免疫细胞和其他一些组织也均有表达 (5,6)。 目前认为 1,25(OH)2D3的免疫调节作用主要是通过其对抗原递呈细胞的作用完 成的,其中最主要的是树突状细胞( dendritic cells,DCs) (7, 8) 。体外试验结果表 明 1,25(OH)2D3能抑制单核细胞或小鼠骨髓来源的前 DCs向成熟 DCs的分化。更为 重要的是,DCs的抗原递呈能力在 1,25(OH)2D3作用下明显下降,表现为其细胞表 面共刺激分子的表达以及白介素 12(IL-12)的产生均下降 (9)。同时,有报道显示 1,25(OH)2D3能促进 DCs产生 IL-10,后者作为免疫抑制细胞因子,可以抑制 Th1细 胞产生 IL-12 (7, 8)。 1,25(OH)2D3对T淋巴细胞的直接作用也有所报道。已证实 1,25(OH)2D3在体外 能抑制T细胞增殖和细胞周期从 G1a期向G1b期的进展。它还能影响T细胞产生细胞 因子,在减少Th1细胞因子IFN-γ产生的同时,增加Th2细胞因子如IL-4, IL-5和IL-10 的产生 (10),从而促进活化的T细胞向Th2细胞的分化。另外,有证据表明 1,25(OH)2D3还能通过下调活化的 T细胞表面的FasL表达,影响T细胞的凋亡 (11)。 7 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , 然而,1,25(OH)2D3对B淋巴细胞功能的影响至今未见有详细的研究。以往的相 关报道也充斥了诸多矛盾的研究结果。一直以来被广泛接受的观点认为, 1,25(OH)2D3对B细胞并没有直接作用,而是通过抑制CD4阳性T细胞的功能或单核细 胞/巨噬细胞产生的细胞因子,间接地对 B细胞产生抑制作用 (12, 13)。 有趣的是,许多自身免疫性疾病患者都报道存在维生素 D缺乏下的现象,其中 包括系统性红斑狼疮 (SLE) (14, 15)。SLE是一种以免疫功能失衡、自身抗体过度产 生为特征自身免疫性疾病 (16)。尽管目前其确切的病因尚不明了,但近年的研究包 括B细胞清除治疗的有效性,强烈提示B细胞可能作为一个中心环节在该疾病的发病 机理中发挥作用(17-20)。由此,我们提出假设,1,25(OH)2D3可能具有维持B细胞稳 态的作用,而1,25(OH)2D3的缺乏可能有助于B细胞的过度激活,参与 SLE的发病。 本研究中,我们检测了SLE患者的25(OH)D3和1,25(OH)2D3血清水平,并分析其 与疾病活动性和自身抗体产生的相关性。其后,我们研究了 1,25(OH)2D3对人外周B 细胞可能存在的直接作用,以期了解其参与自身免疫性疾病发病的更多潜在机制, 并为1,25(OH)2D3应用于B细胞过度活化为特征的疾病如 SLE的治疗前景提供更多切 实依据。 8 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , 第一章 系统性红斑狼疮患者维生素 D水平测定及其临床相关性研究 引言 维生素 D的主要生理学功能是调节机体的钙磷平衡,近年来随着认识的深入, 发现其在免疫功能方面同样起广泛而重要的作用 ( 1)。维生素 D缺乏在许多自身免 疫性疾病患者中有所报道 (14, 15, 30)。 虽然维生素 D可以从食物中获得,但主要的来源是通过皮肤中的光合作用。在 紫外线( 270–300 nm)照射下,皮肤中的 7一脱氢胆固醇转化成维生素 D3进入血液, 在肝内 D3- 25-羟化酶的作用下转化成 25-羟维生素 D3 ( 25( OH) D)3。进一步的羟化则 主要在肾脏近曲小管中完成,在 25( OH) D3- 1-羟化酶 ( CYP27B1)作用下生成具有生 物活性的 1, 25( OH) 2D3(2)。 系统性红斑狼疮(Syst emi c Lupus Er yt hemat osus, SLE)是一种慢性的自身免 疫性疾病,发病原因尚不明确,认为由多因素决定,包括易感基因、环境因素、性 别,以及机体对刺激作出应答时产生的免疫反应及炎症介质的数量。目前还没有治 愈的办法,因此狼疮的发病机理研究及治疗仍具有很大的挑战性。一般认为紫外线 会激发狼疮活动,因此患者通常会被建议避免皮肤直接暴露在强烈的阳光下。由此 我们推测 SLE患者中存在维生素 D缺乏,并因此进一步造成免疫功能的失衡和钙磷 代谢的混乱,参与致病过程。纠正维生素 D缺乏可能成为 SLE治疗中的重要环节。 材料和 9 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , 1.病人来源 112例 SLE患者来自上海仁济医院风湿科门诊和住院病人,诊断均符合 1997 年美国风湿病学会修订的 SLE分类标准(21)。按 SLEDAI积分对患者临床活动度进 行评估(22)。其一般资料详见表 1。其中女性 112例,男性 11例,平均年龄 33.8 ?1.57 (15-66)岁,平均病程 4..52?0.5 (0.01-20)年,平均 SLEDAI积分为 6.5?0.9 (0-22)分。正常对照组 30例,性别年龄构成比与 SLE组无统计学差异。另外类风 湿关节炎患者(RA)对照组 27例,也来自仁济医院风湿科门诊。 研究组与对照组每人采外周静脉枸橼酸抗凝血 3ml,分离血浆待测维生素 D3水 平。 表 1 SLE患者的一般情况 一般情况 SLE患者 N=112 年龄, mean ? SEM( r ange)年 33. 8 ? 1. 57 ( 15- 66) 女性, % 90. 2% ( 101/ 112) 病程, mean ? SEM( r ange)年 4. 52 ? 0. 5 ( 0. 01- 20) SLEDAI积分,平均 ? SEM( r ange) 6. 5 ? 0. 9 ( 0- 22) 关节炎, No ( %) 31. 3% ( 35/ 112) 23. 2% ( 26/ 112) 皮疹, No ( %) 17. 9% ( 20/ 112) 口腔溃疡, No ( %) 37. 5% ( 42/ 112) 肾脏受累, No ( %) 7. 1% ( 8/ 112) 血管炎, No ( %) 25% ( 28/ 112) 血液系统受累, No ( %) 中枢神经系统受累 , No ( %) 3. 6% ( 4/ 112) 糖皮质激素应用, mean ? SEMmg/ day ( r ange) 23. 2 ? 2. 9 ( 0- 160) 2( ELI SA方法检测 25( OH) D3和 1, 25( OH) 2D3 水平 2(1试剂盒 25( OH) D ELI SA试剂盒 ( Alpco Diagnostics, Windham, NH) 10 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , 1, 25( OH) 2D3 ELI SA试剂盒 ( Alpco Diagnostics, Windham, NH) 2(2实验步骤 1)将1000μl的标准样品,阴性对照及血浆样本(血浆或血清)加入抽提管,室温孵育 10分钟。若样品体积小于 1000μl,可相应加入Tr i s- HCl缓冲液,使总容积达 1000μl。 2)用1ml di i sopr opyl et her洗脱维生素D共3次,每次3分钟。使提取液缓慢滴入抽 提管下干燥的硅胶盒柱中。去除抽提管。 3)用2ml异丙醇/己烷(体积比4/ 96)清洗硅胶柱5次 4)用2ml异丙醇/己烷(体积比6/ 94)清洗硅胶柱3次。 5)用2ml异丙醇/己烷(体积比25/ 75)洗脱维生素D,两次。 6)应用真空离心机使抽提液干馏到玻璃管。 7)加入20 ul乙醇到玻璃管,迅速混匀,避免气化。 8)加入450ul抗体溶液到玻璃管,彻底混匀,室温孵育 1小时。 9)加入200ul样品到检测板的每个孔,盖紧,在 4- 8?下孵育18- 22小时。 10)摒弃每孔的液体,每孔加入250ul的稀释缓冲液洗涤 5次。最后一次,用吸水纸清 除残余缓。 11)每孔加入200微升接合液。密闭检测板,放在水平混合机上,室温孵育 1小时。 12)摒弃液体,每孔加入用 250ul稀释缓冲液洗涤5次。每孔加入200ul底物,室温 避光孵育20- 30分钟。 13)每孔加入50ul的中止液,混匀。立刻用ELI SA r eader确定450nm的吸收光谱, 并以620nm作为一种参考。 3(统计分析统计分析采用Microsoft Excel软件,组间均值差异t-检验(双尾,非配 对, Student t检验)。结果以均值 ? SEM表示。符号(*)表示 p值 <0.05, (**) p值 <0.01, (***) p值 <0.001. 结果 11 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , 1( SLE患者的 25(OH)D3水平 如图1所示,SLE患者外周血25( OH) D3水平为11. 5 ? 1. 5 ng/ ml(N=57),显著低 于正常对照组 ( 59. 2 ? 6. 5ng/ ml , N=28)和RA患者组 ( 54. 6 ? 5. 2 ng/ ml , N=27) ( p<0. 001),正常对照组和RA组间无差异( p>0. 05)。 SLE患者组中,有12例为新发病 人,平均发病日期为25. 8 ? 9. 2天,并尚未接受任何免疫抑制治疗,他们的25( OH) D3 水平为11. 6 ? 2. 1 ng/ ml ,同样明显低于正差对照组,而与 45例既往已经诊断并经 过治疗的SLE患者(平均病程为5.11 ? 0.88年)相比(无差异11. 8 ?1. 8 ng/ ml , N=44) ( p>0. 05)。而25( OH)D3的水平与狼疮临床指标包括肾脏受累、SLEDAI积分以及免 疫抑制剂的应用均未发现有相关性。 图 1. SLE患者的 25(OH)D3水平 Figure 1. Patients with SLE have decreased 25(OH)D level. Serum levels of 25(OH)D and 1,25(OH) D levels were measured ELISA. A. 25(OH)D levels in SLE patients (N=57)2 3 compared to normal controls (NC, N=28) and RA patients (N=29). Mean levels in SLE patients were significantly different from those in NC and RA patients (P<0.001). B. 25(OH)D levels in patients with established SLE with a mean duration since diagnosis of 5.11 ? 0.88 years (N=45) compared to newly diagnosed, untreated patients with a mean duration of 25.8 ? 9.2 days (N=12). There was no significant difference in mean 25(OH)D levels in newly diagnosed or established SLE patients (p>0.05). 2( SLE患者的1,25(OH)2D3水平 明显低于正常对照 SLE患者1,25(OH)2D3水平为14. 5 ? 1. 2 pg/ ml ( N=87) , 组 ( 29. 8 ?1. 5 pg/ ml , N=30) ( p<0. 001) (图2A)。用SLEDAI积分对SLE患者进行分 组,在SLEDAI积分>4的患者组中,1, 25( OH) 2D3水平为12.2 ?1.6 ng/ml (N=52 ),而 3 SLEDAI score ? 4的患者中,1, 25( OH) 2D水平为19.4 ? 1.9 ng/ml (N=36) (图2B), 12 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , 前者明显低于后者(P<0.001)。对SLE患者的抗体检测结果进行分析,结果发现有 血清检测 ANA阳性的患者 1,25(OH)2D3 水平明显低于 ANA阴性患者 (14.4 ? 1.3 pg/ml, N=71 versus 22.0 ? 4.2 pg/ml, N=11, p<0.05),而dsDNA抗体阳性组和阴性 组间则无差别 (15.0 ?1.8 pg/ml, N=39 versus 13.5 ? 1.7 pg/ml, N=43, p>0.05) (图 3)。以上结果显示1,25(OH)2D3水平在SLE患者中明显低下,且与临床活动度以及 ANA的产生明显相关。 图2 SLE患者1,25(OH)2D3水平检测及临床相关性 SLE activity *** 50 30 40 *** 30 20 20 10 10 0 0 NC SLE non-active active N=30 N=58 N=25 N=33 ANA 25 ds-DNA * 20 20 15 15 10 10 5 5 0 0 positive negative positive negative N=19 N=10 N=10 N=19 Figure 2 Patients with SLE have decreased 1,25(OH)2D levels. A. 1,25(OH)2D levels in 3 3 SLE patients (N=87) compared to normal controls (NC) (N=30). The 1,25(OH) D levels were 2 3 significantly lower in SLE patients (P<0.001). B. 1,25(OH) D levels in non-active SLE 2 3 patients (SLEDAI ?4, N=35) compared to active patients (SLEDAI >4,N=52). Active SLE patients had significantly lower levels of 1,25(OH) D (P<0.001). C. 1,25(OH) D levels in SLE 2 3 2 3 patients with positive ANA (N=71) compared to those without ANA (N=11). Patients without ANA had significantly higher levels of1,25(OH) D (P<0.05). D. 1,25(OH) D levels in SLE 2 3 2 3 patients with anti-dsDNA (N=39) compared to those without anti-dsDNA (N=43). 1,25(OH)2D 3 13 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , levels did not differ between these groups. Data are shown as mean ? SEM and significant differences between the groups are shown by *** (p<0.001) or * (p<0.05). 讨论 维生素D缺乏在许多自身免疫性疾病患者中有所报道,包括多发性硬化、类风 湿关节炎(RA), 1型糖尿病,纤维肌痛症以及 SLE (14, 15, 30)。SLE是以免疫系统紊 乱,抗核抗体(ANA)产生、免疫复合物形成、补体系统激活为特征的自身免疫性疾 病(16)。 相对于1,25(OH)2D3,由于25( OH) D3半衰期较长为2周,且不受钙、磷和甲状旁 腺素(PTH)的直接调节,故认为其血浓度能更好代表机体维生素 D的营养状况。1997 年美国食品和营养委员会确定血浆 25( OH) D水平为检测体内维生素D营养状况的有效 指标。我们对中国狼疮患者的 25( OH) D3的血浆浓度进行了检测,结果证实其水平显 著低于健康对照组和RA患者组。此外,我们还发现即使是在12名初发尚未接受任何 治疗的狼疮患者中,其25(OH)D3水平也明显低于对照组,和已明确诊断并接收免疫 抑制剂治疗的患者无差异,由此提示维生素 D的缺乏可能在患者临床发病之前就已 经存在,甚至参与了狼疮发病。已知非洲裔美国人通常存在慢性维生素 D缺乏(29, 49),可能与非裔人种对奶制品不耐受、皮肤色素多和光照减少有关。而 SLE在这一 人群中的发病率和严重度也高于其他人群 (50),这两种疾病在流行病学上的联系也 许由此可以得到部分解释。与文献报道不同,本研究中未发现 RA患者25(OH)D3水 平降低。 同时,我们对狼疮患者维生素 D活性成分,1,25(OH)2D3,的血浆浓度进行了检 测,结果发现狼疮患者同样存在1,25(OH)2D3水平低下,且还与疾病活动度以及ANA 的产生负相关。由于样本有限,对这12名初发病人未能检测1,25(OH)2D3水平,因而 尚不能确定1,25(OH)2D3的低下同样存在于新发病人中。 已知SLE患者在临床发病之前机体可能已经存在免疫学异常,近年的一项大样 本回顾性研究就证明SLE患者在临床诊断前可以存在自身抗体长达数年 (51),那么极 有可能维生素D缺乏同样参与这一过程。由此我们推测,维生素 D参与正常B细胞稳 14 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , 态的维持,而SLE患者中的维生素D缺乏则有助于B细胞过度活化、耐受失衡以及产 生致病性的自身抗体。上述结果中1,25(OH)2D3水平降低与ANA产生有相关性,就 支持这一可能性。 本研究中,约有 13.4%(11/82)SLE患者免疫学检测呈 ANA阴性,与ANA阳性的 患者相比,他们的平均1,25(OH)2D3水平相对较高,两组间有显著性差异。然而,这 种差异在dsDNA阳性和阴性组之间却未观察到。有研究者认为, SLE患者存在的持 续并对治疗耐受的ANA,是来源于一类特殊的长期存活的浆细胞,它们可以在没有 记忆B细胞的情况下产生抗体介导的自身免疫记忆 (52)。而与此不同,dsDNA抗体 通常与狼疮的活动性相关,通过免疫抑制治疗或 B细胞清除可以减少其产生,被认 很可 为是来源于短期存活的浆母细胞(plasmablasts)(20, 53)。因此,1,25(OH)2D3 能在调节长期存活的浆细胞产生中起一定作用,而对抑制分裂中的浆母细胞产生抗 体作用相对较弱。 因此我们相信,1,25(OH)2D3参与了B细胞的稳态维持以及浆细胞的分化调节, 为验证这一观点,我们在本课题的第二部分对1,25(OH)2D3对B细胞的一系列作用进 行研究。 15 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 第二章 1,25二羟维生素 D3对人外周 B淋巴细胞的免疫调节作用及其机理 研究 引 言 1,25-二羟维生素D3 (1,25(OH)2D3)是维生素D3的生物活性成分,自1923年发现 以来一直用于佝偻病的治疗,在维持骨骼和钙磷代谢中起重要作用。近年来的研究 显示1,25(OH)2D3还具有广泛的免疫调节作用 (1)。虽然维生素D3可以从食物中获得, 但主要的来源是通过皮肤中的光合作用。在紫外线 (270–300 nm)照射下,皮肤中的7 一脱氢胆固醇转化成维生素 D3进入血液,在肝内D3-25-羟化酶的作用下转化成 25- 羟维生素D3 (25(OH)D3)。进一步的羟化则在肾脏近曲小管中完成,在 25(OH)D3-1- 羟化酶 (CYP27B1)作用下生成具有生物活性的 1,25(OH)2D3。24-羟化酶 (CYP24A1) 是其主要的降解酶,可以在 1,25(OH)2D3诱导下产生 (2)。在某些情况下,肾外其他 部位的组织细胞,如活化的巨噬细胞和树突状细胞也能表达 CYP27B1,但这种转变 较肝肾通路含量甚微,目前对这种转变的生理意义尚不清楚,有可能起自分泌或旁 分泌的作用(3, 4)。已知的1,25(OH)2D3生物学效应主要是通过维生素 D受体(VDR)介 导,现已证明VDR作为核激素受体超家族的一员,不仅存在于经典的靶组织 (如小 肠、骨、肾脏、甲状旁腺 ),各种免疫细胞和其他一些组织也均有表达 (5,6)。 目前认为 1,25(OH)2D3的免疫调节作用主要是通过其对抗原递呈细胞的作用完 成的,其中最主要的是树突状细胞( dendritic cells,DCs) (7, 8) 。体外试验结果表 明 1,25(OH)2D3能抑制单核细胞或小鼠骨髓来源的前 DCs向成熟 DCs的分化。更为 重要的是,DCs的抗原递呈能力在 1,25(OH)2D3作用下明显下降,表现为其细胞表 面共刺激分子的表达以及白介素 12(IL-12)的产生均下降 (9)。同时,有报道显示 1,25(OH)2D3能促进 DCs产生 IL-10,后者作为免疫抑制细胞因子,可以抑制 Th1细 胞产生 IL-12 (7, 8)。1,25(OH)2D3对 T淋巴细胞的直接作用也有所报道。已证实 1,25(OH)2D3在体外能抑制 T细胞增殖和细胞周期从 G1a期向 G1b期的进展。它还 影响 T细胞产生细胞因子,在减少 Th1细胞因子 IFN-γ产生的同时,增加 Th2细胞 16 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 因子如 IL-4, IL-5和 IL-10的产生 (10),从而促进活化的 T细胞向 Th2细胞的分化。 另外,有证据表明 1,25(OH)2D3还能通过下调活化的 T细胞表面的 FasL表达,影响 T细胞的凋亡 (11)。 然而,1,25(OH)2D3对B淋巴细胞功能的影响至今未见有详细的研究。以往的相 关报道也充斥了诸多矛盾的研究结果。一直以来被广泛接受的观点认为, 1,25(OH)2D3对B细胞并没有直接作用,而是通过抑制CD4阳性T细胞的功能或单核细 胞/巨噬细胞产生的细胞因子,间接地对 B细胞产生抑制作用 (12, 13)。 通过对 SLE患者的 1,25(OH)2D3血浆水平的检测,我们发现 SLE 患者的 1,25(OH)2D3水平明显低于健康人群,并且与疾病活动度、抗核抗体的产生负相关。 SLE是一种以免疫功能失衡、自身抗体过度产生为特征自身免疫性疾病 (16)。近年 的研究包括B细胞清除治疗的有效性,提示B细胞可能作为一个中心环节在该疾病 的发病机理中发挥作用(17-20)。由此,我们提出假设,1,25(OH)2D3可能具有维持B 细胞稳态的作用,而1,25(OH)2D3的缺乏可能有助于B细胞的过度激活,参与SLE的 发病。为验证这一可能性,我们对 1,25(OH)2D3对健康人外周血B细胞的作用及其潜 在的机理进行探索。 材料和方法 1.材料 1.1试验仪器 低温高速离心机 (Beckman) (Millipore) Milli-Q plus 二氧化碳培养箱 (BIO-RAD) four-color FACScalibur (Becton Dickinson) Dako Cytomation MoFlo (Dakocytomation, Fort Collins, CO ) 微定量盘式闪烁计数仪 (Packard Instruments, Downers Grove, IL) Uni Fi l t er 96-孔收集器 ( Per l i nEl mer , Waltham, Massachusetts, USA) 96-孔滤紙內建式定量板 ( Uni Fi l t er : f i l t er bui l t - i n mi cr opl at e) 17 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , ND1000紫外/可见分光光度计 ( NanoDr op,US) Applied Biosystems 7700 PCR仪 (Applied Biosystems) 酶标仪 (Bio-Rad550) 96孔聚苯乙烯塑料板 (Nalge Nunc International, Rochester, NY) ELISpot板:MultiScreen-HTS plates (PVDF membrane, Millipore, Bedford, MA) 分析软件:FlowJo software (TreeStar, Stanford University, Palo Alto, CA). 1.2试剂 RosetteSep? Human B Cell Enrichment Cocktail (Stem Cell Technology, Vancouver, Canada). 细胞凋亡检测试剂盒: Annexin V Kits,Oncogene (San Diego, CA, USA) 碱性磷酸酶标抗的羊抗人 I gG或 I gM( Bet hyl Labor at or i es ) pNPP碱性磷酸底物片 (Sigma, St. Louis, MO) goat anti-human IgG and IgM (Bethyl Laboratories, Montgomery, TX) carbonate phosphate-buffered saline (Sigma) alkaline phosphatase substrate Kit III (Vector Laboratories, Inc. Burlingame, CA) biotinylated goat–anti-human IgM or IgG (Invitrogen) alkaline phosphatase-conjugated streptavidine (Sigma) Cellular Technology Ltd Series 3B Analyzer (CTL Analyzer LCC, Cleveland, OH) 总RNA提取试剂盒:Rneasy Mi ni Ki t ( QI AGEN, Val enci a, CA) SuperScript One-Step PCR System with platinum Taq polymerase and ROX reference dye (Invitrogen Life Technologies). TaqMan引物 / 探针 (Applied Biosystems (Foster City, CA): β2M (beta-2-microglobulin, Hs99999907_m1), VDR (Hs01045840_m1), CYP27B1 (Hs00168017_m1), CYP24A1 (Hs00167999_m1), Pax5 (paired box gene 5, Hs00277134_m1), Bcl6 (B-cell CLL/lymphoma 6 (zinc finger protein 51, Hs00153368_m1), Blimp1( B-lymphocyte-induced maturation protein 1, Hs00153357_m1), IRF4 (interferon regulatory factor 4, Hs00180031_m1), XBP1 18 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , (X-box binding protein 1, Hs00231936_m1), ERN1 (endoplasmic reticulum to nucleus signaling 1, Hs00176385_m1), AID (activation-induced cytidine deaminase, Hs00221068_m1), p27 (cyclin-dependent kinase inhibitor 1B, Hs00153277_m1), p21 (cyclin-dependent kinase inhibitor 1A, Hs00355782_m1) and p18 (cyclin-dependent kinase inhibitor 2C, Hs00176227_m1) Human Cell Cycle RT?Profiler? PCR Array (SuperArray Bioscience, MD) Human NFκB Signaling Pathway RT?Profiler? PCR Array (SuperArray ) PT2 PCR array first strand kit (SuperArray Bioscience) RT Real-Time SYBR Green/ROX PCR Master Mix (SuperArray Bioscience) 细胞因子和刺激剂:anti-CD40 (R&D Systems, Minneapolis, MN), goat F(ab’)2 anti-IgM (Jackson ImmunoReseach, West Grove, PA), IL-21 (Cell Sciences, Canton, MA), IL-4 (R&D Systems, Minneapolis, MN), 1,25(OH)2D3 (usually 10 nM) and 25(OH) D3 ( Sigma, Saint Louis, MO) 单克隆荧光标记抗体: anti-IgD-FITC, anti-CD27-PE, anti-CD40-PE, anti-CD86-FITC, anti-IgG-PE, anti-CD19-APC, anti-HLA-DR-PE, (Becton Dickinson Pharminogen, San Diego, CA), anti-CD19-PerCpCy5.5, anti-CD38-APC (clone HB7) (Becton Dickinson Immunocytometry, San Jose, CA) 细胞培养液:10,FCS RPMI1640 染色缓冲液:1%BSA, 5%FCS/ PBS 1.3标本来源 健康者的外周新鲜全血或 Buffycoat来自 Warren G. Magnuson临床中心血库。标本 收集经 National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institute of Diabetes & Digestive & Kidney Diseases (NIAMS/NIDDK)评估委员会批 准,同时根据 Helsinki声明要求获得书面知情同意书。 2.实验方法 2.1 B细胞分选和纯化 1).新鲜EDTA抗凝全血10ml或Buffycoat 10ml, Add at 0.5ml抗体,充分混匀,20 19 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 分钟。 2).全血标本用形同体积 PBS+ 2% FBS稀释,混匀。 3).在15ml离心管中置入6ml Ficoll,小心地将9ml标本用滴管沿管壁缓慢叠加于分 层液面上,注意保持清楚的界面。室温下水平离心 600g×20分钟。 4).离心后管内分为三层,上层为血浆和PBS液,下层主要为红细胞和粒细胞。中层 为淋巴细胞分离液,在上、中层界面处有一以靶细胞为主的白色云雾层狭窄带。。用 毛细吸管插到云雾层,吸取靶细胞。置入 15ml离心管中,加入10ml RPMI1640, 1500rpm×10分钟,洗涤细胞两次 5).末次离心后,弃上清,加入细胞培养液,重悬细胞。 6). Anti-CD19直接免疫荧光染色 ,检查B细胞纯度,一般在 90-92%以上。 2.2 B细胞体外培养和激活 1)(纯化的 B细胞用培养液(10% FCS/ RPMI,1%L-glutamine, 1%青霉素-链霉素) 调整为终浓度为 1x106 /ml 2)(96孔 U型细胞培养板,每孔加 100 µl细胞悬浮液 3).根据实验要求加入激活剂和 /细胞因子: 1 µg/ml anti-CD40, 3 µg/ml goat F(ab’)2 anti-IgM, 250 ng/ml IL-21, 50 ng/ml IL-4, 1,25(OH)2D3 (usually 10 nM) and 25(OH) D3 4)(置入 37?、5,CO2温育箱中孵育 2.3流式细胞术检测时的样品制备 细胞直接免疫荧光标记法 1) 1).将纯化的 B细胞用细胞培养液调整细胞浓度为 2×107/ml 2)取 5µl细胞悬液加入预先有特异性 McAb(0.5,2µl)的 96孔 U型板中,充分混 匀,4?,避光 20min 3) 3. 2%FCS/PBS洗涤液洗涤 2次,每次加洗涤液 200ul左右 ,4? 1500rpm×5min 4) 4.弃上清,加入 200ul固定液(1% paraformaldehye/洗涤液),充分混匀 20 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 5) 5.立即上机或 4?避光保存(24小时内检测) 2. 4 Annexi n V/ PI双染色法检测细胞凋亡 1)细胞收集:将培养的 B细胞直接收集到 2ml的离心管中,调整细胞浓度为 1×106/ml, 1500r/min离心 5min,弃去培养液。 2)用 2%FCS/PBS洗涤 1次,1500r/min离心 5min。 3)用 100ul的标记溶液重悬细胞,加入 FITC- Annexin V(终浓度 1ug/ml),室温下避 光孵育 10~15min。 4) 1500r/min离心 5min沉淀细胞孵育缓冲液洗 1次。 5)加入 PI溶液,4?避光,孵育 20min,避光并不时振动。 6)流式细胞仪分析:流式细胞仪激发光波长用 488nm,用一波长为 515nm的通带 滤器检测 FITC荧光,另一波长大于 560nm的滤器检测 PI。 7)结果判断:凋亡细胞对所有用于细胞活性鉴定的染料如 PI有抗染性,坏死细胞 则不能。细胞膜有损伤的细胞的 DNA可被 PI着染,而细胞膜保持完好的细胞 则不会。因此,在细胞凋亡的早期 PI不会着染。正常活细胞与此相似。在双变 量流式细胞仪的散点图上,左下象限显示活细胞,为(Annexin V-/PI-);右上象 限是非活细胞,即坏死细胞,为(Annexin +/PI+);而左上象限为凋亡细胞,显 现(Annexin +/PI-)。 2.5细胞增生测定 2.5.1 3H-胸腺嘧啶核苷掺入法: 1) B细胞培养至第 3,4或 5天,每孔加入 3H-胸腺嘧啶核苷 1µCi。 2)继续孵育 16小时 3)用 ZT-?型微量细胞收集仪将 96-孔培养板內之的細胞收集至 96-孔滤紙內建式 定量板内,每孔加入 25μ,的二甲苯闪烁计数液,置入液体闪烁计数仪內計测定 3 H-胸腺嘧啶核苷掺入每分钟计数值 ( count s per mi nut e,cpm) CFSE标记法 2.5.2 1)纯化的 B细胞用 PBS洗涤两次,悬浮于 PBS溶液中,调整细胞浓度为 2×107/m 2) CFSE标记染色,终浓度 1 µM,充分混匀,室温下 15分钟 21 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 3)加入相同体积的 100% FCS终止标记,静置 2分钟 4)细胞培养液洗涤 2次,通过流式细胞仪记录起始 CFSE荧光强度。 5)细胞培养 3至 6天后,再次记录 CFSE荧光强度. 2.6 ELISA法测定免疫球蛋白 1).包被:用 PBS将鼠抗人 IgG或 IgM抗体 1:500稀释。在每个聚苯乙烯板的反 应孔中加 50ul,4?过夜。次日,弃去孔内溶液,用洗涤液 (0.05%Tween20/TBS)冲 液洗 3次,每次 3分钟。(简称洗涤,下同)。 2).阻断:每孔中加 200ul阻断液(1%BSA/PBS),室温下至少 1小时。 3).加样:加一定稀释的待检样品 50ul于上述已包被之反应孔中,置 4 ?过夜或 室温 3小时。然后洗涤。 (同时做空白孔,阴性对照孔及标准浓度样品 )。 4).加酶标抗体:于各反应孔中,加入新鲜 1:5000稀释的碱性磷酸酶标抗的羊抗 人 IgG或 IgM 50ul。置 4?过夜或室温 3小时。然后洗涤。 5).加底物液显色:于各反应孔中加入新鲜配制的 pNPP碱性磷酸底物溶液 50ul, 避光,室温 10,30分钟。 6).结果判定: ELISA检测仪测 OD值(410nm处),以空白对照孔调零后测各孔 OD值。 . 2. 7 ELI SPOT方法检测I g抗体分泌的B细胞 1) ELISpot板每孔加入无菌PBS(pH 7.4)200ul,室温下1小时,湿润PVDF膜, 弃除PBS。 2)每孔加入50ul包被抗体溶液,室温3小时。甩掉多余抗体,用无菌PBS洗板3次, 200ul/孔。加入阻断液200ul,室温1小时。甩干。 3)收集培养板中的B细胞,无菌PBS洗涤3次,重新悬浮于培养液中,稀释成不同 浓度(5X105 , 1X105,5X104,1X104),加入ELISpot板中,每孔200ul。 4)将培养板放入37?、5, CO2孵箱内孵育24小时。 5)甩掉细胞,用TBS/0.05%Tween 20洗板4次,200ul/孔。 6)加入1:2000稀释后的生物素化特异性检测抗体。每孔 50ul,室温孵育3小时。 22 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 7)甩掉多余抗体,用TBS/0.05%Tween 20洗板4次,200ul/孔 8)用TBS/0.05%Tween稀释ALP标记链亲和素,0.5ug/ml, 100ul/孔,室温孵育1小 时。 9)甩掉多余液体,洗板 4次,200ul/孔。 每孔加入100ul ALP显色底物溶液,室温下,至出现清晰斑点。 10) H2O冲洗终止反应。将板晾干。 ELISpot读板仪计数斑点个数。 . 2.8 RNA制备及定量RT-PCR. 2.8.1 RNA制备 1)收集新鲜纯化的或体外培养后的 B细胞,PBS洗涤一次。 2)加入350μl Buf f er RLT(含10ug/ ml B- ME),并充分混匀。加入过滤柱,10000r / mi n 离心洗涤2分钟,弃去柱子。 3)加入350μl无水乙醇,Ti p头充分混匀。 4)将共计700μl含总RNA的溶液转入套在2 ml离心管内的RNeasy柱子内, 10000r / mi n离心15秒,弃去滤过液。 5)吸取700μl Buf f er RW1到RNeasy mi ni柱子内,10000r / mi n离心15秒,弃去滤 过液及套管。 6)吸取500μl Buf f er RPE到RNeasy mi ni柱子内,10000r / mi n离心15秒,弃去滤 过液,再用500μl Buf f er RPE在10000r / mi n离心洗涤2 mi n,弃去滤过液和2 ml 的套管,将RNeasy mi ni柱子转入一新的1. 5 ml Eppendor f管中。 7)吸取 30μl RNase f r ee的水,10000r / mi n离心洗脱1 mi n。 8) NanoDr op分光光度计测定RNA浓度及纯度. 2.8.2 RT-PCR :采用 SuperScript One-Step PCR System with platinum Taq polymerase RT-PCR 2×buffer: 12.5 ul Mg2SO4(50nM) 0.6ul (1.2nM) RNA 5uL( 50ng) 23 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , PRIMER/probe 1ul RT/platinum Taqmix 0.5ul ROX dye 0.25ul H2O 5.15ul 25ul PCR条件: cDNA合成:46?×30min 第一次变性:94?×2min 变性:95?×15sec 退火:55?×30sec 共 40个循环 延长:72?×1min .最后一次延长:72?×15min。 2.9统计 统计分析采用 Microsoft Excel软件,组间均值差异 t-检验(双尾 ,非配对或配对 , Student t检验)。结果以均值 ? SEM表示。符号 (*)表示 p值 <0.05, (**) p值 <0.01, (***) p值 <0.001. 结果 1. 1,25(OH)2D3抑制人外周血B淋巴细胞的增生 首先,我们对 1,25(OH)2D3对 B细胞存活的影响进行了检测。如图 1A所示, 未激活的 B细胞存活率不受.1,25(OH)2D3影响。接着,我们观察了 1,25(OH)2D3对 B 细胞增生的作用。3H-胸腺嘧啶核苷掺入法测定结果显示, 1,25(OH)2D3对 B细胞 增生有明显的抑制作用,这种效应在 B细胞受到多种刺激剂共同作用时尤为明显, 如 anti-IgM/anti-CD40 (p<0.05), anti-CD40/IL-21或 anti-IgM/anti-CD40/IL-21 (p<0.01)。当单用 anti-CD40或 anti-IgM刺激 B细胞时,由于 B细胞所获得的增生 能力有限,因而这一抑制效应相对不明显,有时缺乏重复性(图 1B )。同时,应用 CFSE标记法我们观察了 1,25(OH)2D3抑制 B细胞增生的时间效应。如图 1C所示, B细胞体外试验培养 3天后,可以明显看到细胞中标记的 CFSE荧光强度开始稀释, 这时 1,25(OH)2D3处理的 B细胞组与对照组之间尚未见明显的差异。随着时间延长, CFSE荧光强度进一步稀释,而 1,25(OH)2D3处理的 B细胞中高稀释度的细胞数量 24 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 则明显少于对照组。这些结果显示 1,25(OH)2D3 主要抑制增生中的 B细胞进一步增 生,而非最初的细胞分裂。 图1. 1,25(OH)2D3对人外周血B淋巴细胞增生的影响 300 nil C. VitD A. 200 60 day 3 nil 100 VitD 40 0 20 300 0 200 day 4 day 1 day 2 day 3 100 B. 0 300 day 5 200 1 6 0 0 0 nil 1 2 0 0 0 100 VitD ** 8 0 0 0 ** 0 4 0 0 0 * 300 0 * * 200 n il day 6 100 0 CFSE Figure 1. Vitamin D inhibits proliferation of human peripheral B cells. A. Purified B 2 3 cells were cultured with or without 1,25(OH) D (10 nM) and stained with Annexin V /PI. Percentages of viable cells (double negative for Annexin V and PI) are shown as mean ? SEM of triplicate cultures and are representative of three independent experiments. B. Purified B cells were stimulated with combinations of anti-IgM, anti-CD40 and IL-21 as indicated in the presence or absence of 1,25(OH) D (10nM). After 3 days, cells were pulsed with 3H-thymidine and 2 3 incorporation was measured 16 h later. Data are the mean ? SEM of triplicate cultures and are representative of three independent experiments. Significant differences between Vitamin D and control responses are shown by * (p<0.05) or ** (p<0.01). C. Purified B cells were labeled with CFSE and cultured with anti-IgM, 2 3 anti-CD40 and IL-21 in the presence or absence of 1,25(OH) D (10 nM). At the time-points indicated, CD19+ B cells were analyzed for CFSE dilution. Data are representative of results from three experiments. 25 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 2. 1,25(OH)2D3能诱导外周血B淋巴细胞的凋亡 为进一步了解 1,25(OH)2D3对 B细胞增生的抑制效应是否与其诱导凋亡有关, 我们应用 Annexin V/PI双染色法比较了 1,25(OH)2D3处理组与对照组间 B细胞发生 早期凋亡的比例。结果如图 2A所示,B细胞经过 4天的体外培养,在没有任何刺 激剂的条件下,早期凋亡的细胞比例在 1,25(OH)2D3组和对照组中分别为 64.7%和 62.5%,两组间无差异。而当 B细胞在 anti-IgM/anti-CD40作用下激活时,早期凋 亡的细胞比例明显下降,然而, 1,25(OH)2D3处理组中的凋亡细胞比例则显著高于 对照组 (27.5% vs. 37.5%, p<0.01)。这一现象有时间依赖性,仅见于第 4天(图 2B)。 图2. 1,25(OH)2D3对外周血B淋巴细胞凋亡的影响 B. nil, nil nil, VitD 80 60 40 ** 20 0 day 3 day 4 Figure 2. Vitamin D induces apoptosis of activated human peripheral B cells. Purified B cells were cultured with no stimulus, anti-IgM and anti-CD40 alone or 2 3 combination in the presence or absence of 1,25(OH) D (10 nM). A. After 4 days in culture, cells were washed and stained with Annexin V and PI and analyzed by flow cytometry. The numbers in the upper left quadrants represent the percentage of apoptotic cells in culture. B. Percentages of apoptotic cells in 3 and 4-day cultures with no stimulus or anti-IgM and anti-CD40 and with or without addition of 2 3 1,25(OH) D (10 nM). Data are mean ? SEM of triplicate determinations from one of three independent experiments. Asterisks indicate the statistical significance of the difference between Vitamin D and untreated B cells (p?0.01). 3.激活的 B淋巴细胞能上调表达 1,25(OH)2D3反应基因 接着,应用rt-PCR检测了维生素D受体 (VDR)和已知的1,25(OH)2D3反应基因, 24-羟化酶 (CYP24A1)在B细胞上的表达。结果如图 3A,新鲜分离的B细胞能结构性 的低水平表达VDR mRNA。经过体外3天培养,激活的B细胞表达VDR水平明显上 升,平均增加倍数在anti-IgM/anti-CD40, anti-CD40/IL21, anti-IgM/anti-CD40/IL21条 件下分别为1.45, 2.88和 4.44。而没有任何刺激剂的 B细胞同样能上调1.46倍。无论 26 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 是激活还是非激活的 B细胞,VDR mRNA的表达都能在1,25(OH)2D3作用下进一步 上调。与VDR的表达有所不同,无论在激活还是静息 B细胞中都无法检测到 CYP24A1的表达。而1,25(OH)2D3能显著诱导其在 B细胞中的表达(图3B)。这一 结果证明人外周 B细胞表达 VDR,在激活信号或 1,25(OH)2D3作用下其表达可以上 调,而 CYP24A1的表达则仅受1,25(OH)2D3作用诱导。 图 3. VDR和 CYP24A1在 B淋巴细胞上的表达 Figure 3. Vitamin D inducible genes are upregulated on activated B cells. Purified B cells were stimulated in various conditions in the presence or absence of 2 3 1,25(OH) D (10 nM) as indicated. Total RNA was extracted from cells at day 0 and after 3 days in culture. Gene expression was detected by quantitative RT-PCR in triplicate. A. Expression of VDR relative to β2M. Left panel shows results from stimulated versus fresh B cells, and the right panel shows results from B cells culture without stimulation. B. Expression of CYP24A1 relative to β2M. Left panel shows results from stimulated versus fresh B cells, and the right panel shows results from B cells cultured without stimulation. Data are the mean ? SEM from triplicates from one of two representative experiments. Significant differences between Vitamin D and control responses are shown by * (p<0.05) , ** (p<0.01) or *** (p<0.001). 27 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 4. 1,25(OH)2D3能使激活的外周血 B淋巴细胞表面分子 CD38表达增高 以往曾有报道,1,25(OH)2D3能作用于淋巴细胞增高 CD38表面分子的表达 ( 23, 24)。为进一步验证这一结果,同时了解 1,25(OH)2D3是否还对其它 B细胞表面标 志性分子有影响,我们在纯化的 B细胞体外培养液中加入不同的刺激剂,比较了一 系列表面分子在 1,25(OH)2D3处理的 B细胞与对照之间的差异。其中,CD38在激 活的 B细胞表面的表达受 1,25(OH)2D3影响明显增高,而这种差异在未激活的或用 anti-CD40或 anti-IgM单独刺激的 B细胞上则不明显。相对应的,其他分子的表达 如 IgD, CD21, CD23, CD27, CD86以及 HLA-DR等虽然在刺激剂作用下有所改变, 但 1,25(OH)2D3 却未见对其有影响(图 4A)。为检测 1,25(OH)2D3对 B细胞 CD40表 达的影响,我们应用 ant i - I gM和 I L- 4来激活 B细胞冰上调其表面 CD40的表达, 结果未见 1,25(OH)2D3对 CD40的表达有影响。这一结果显示 1,25(OH)2D3特异性地 增加激活的 B细胞表达 CD38的水平,但对其他一系列分化、激活的标记分子没有 作用。 高表达的 CD38常被视为浆细胞的标记型分子 ( 25)。我们已知 1,25(OH)2D3能 上调 CD38 表达,为明确这一作用是否与浆细胞分化相关,我们应用已知的能诱导 外周 B细胞分化为浆细胞的实验体系 ( ant i - CD40/ I L- 21或 ant i - I gM/ ant i - CD40/ I L- 21)来检测 1,25(OH)2D3对浆细胞分化的作用 ( 26)。结果显示 1,25(OH)2D3作用下的 CD38表达上调与 CD27的表达上调并无相关性。事实上, 1,25(OH)2D3处理组中产生的浆细胞 ( CD38hi , CD27hi )比例明显少于对照组 (图 4B)。CD38表达上调和浆细胞产生减少的作用都随 1,25(OH)2D3浓度增加而增加,其 作用高峰均出现于为 10 nM甚至更少时 (图 4C)。 28 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 体外培养的B细胞在ant i - CD40/ I L- 21或 ant i - I gM/ ant i - CD40/ I L- 21作用下,从第 五天可见浆细胞分化,以后该比例逐渐增加。 1,25(OH)2D3则明显减少培养细胞中 定义为浆细胞的实验中,这一 浆细胞的比例。总的来说,以CD19+/- CD38hi CD27hi 抑制作用平均可达 23-34% (图5A)。 同时,对B细胞培养液中免疫球蛋白含量分析,显示1,25(OH)2D3显著减少了Ig 的分泌。体外培养的B细胞在ant i - CD40/ I L- 21或 ant i - I gM/ ant i - CD40/ I L- 21作用 下,从第5天起培养液中可检测到I g,其含量随着时间的延长而增多(图5B)。这一过 程中,1,25(OH)2D3显著减少了IgG的分泌量,其抑制作用始见于第 5天,以后逐渐 增加直至第9天最为明显(p<0.001)。然而,1,25(OH)2D3对IgM产生的抑制作用则相 对较弱,且仅见于第 9天。通过ELISPOT方法,我们还对分泌Ig的B细胞数进行了计 数,也得到了相一致的结果。1,25(OH)2D3 显著减少能分泌 Ig的B细胞数量 (图 5C)。 值得注意的事,如果在 B细胞体外培养第5天再加入 1,25(OH)2D3,在以后的5 天观察中,则无法见到1,25(OH)2D3对浆细胞和Ig产生的抑制作用(图5D)。这一结果 提示1,25(OH)2D3 能抑制浆细胞产生,而对已经产生的浆细胞则没有作用。 30 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 图 5. 1,25(OH)2D3对浆细胞分化以及免疫球蛋白产生的影响 100 A. 25 nil 75 20 VitD * 15 * 50 10 5 25 0 day 5 day 7 day 9 day 5 day 7 day 9 0 αCD40/αIL21 αIgM/αCD40/IL21 αCaCD40/IL21D40/αIL21 αIaIgM/aCD40/IL21gM/αCD40/αIL21 NN=12=12 N=8N=8 B. αCD40/αIL21 αIgM/αCD40/IL21 αCD40/IL21 2 5 25 *** 25 Nil 2 0 Nil Nil VitD 20 20 * VitD *** 1 5 * VitD 15 15 1 0 10 10 * * 5 5 5 ** 0 0 0 day 5 day 7 day 9 day 5 day 7 day 9 day 5 day 7 day 9 αCD40/IL21 αIgM/αCD40/IL21 αCD40/IL21 C. Nil VitD Nil VitD Nil VitD Q kTi me?andc a nd a ui TI FF e( Un nc m es ss e d ) tdesi o ictpurrm es. so r are TIneF Fe ecee tomhsip pcituroer a r ede e prd to ee h p e QdmeduiprkteTcosisseem?de)antddhesiacoQdmeduiprkteTcosisseem?de)antddhesiaco QdmeudpirkteTcosisseem?de)antddhesicaQdmeudpirkteTcosis em?de)antddhesica TI FaFr e( Unneceo mcitpurrees.sor TI FaFr o mcitpurrees.sor TI FaFr e(Unecno omcitpurr es.sor TI FaFr e ecno omcitpurrees.sor . p p e p e p e( Unnece e(Un IgG IgM 16000 n il nil 2000 VitD VitD 12000 1500 *** 1000 8000 *** ** 500 4000 0 0 αCD40/αIL21 αIgM/αCD40/αIL21 αCD40/αIL21 αCD40/IL21 αCD40/IL21 αCD40/IL21 D. 8 nil 25 nil 25 VitD nil 6 V itD 20 20 VitD 15 15 4 * . 10 10 2 5 5 0 0 0 day 8 day 10 day 8 day 10 day 8 day 10 Figure 5. The inhibitory effect of Vitamin D on plasma cell differentiation and Ig production. Purified B cells were cultured with anti-CD40 and IL-21 with or without anti-IgM in the presence or absence of 1,25(OH) D (10 nM). A. The generation of plasma cells (CD19+/-, 2 3 CD27hi, CD38hi) was assessed after 5, 7 and 9 days in culture (left panel). The frequencies of plasma cells in Vitamin D versus control cultures after a 7 day incubation of anti-CD40/IL-21 (n=12) and anti-IgM/anti-CD40/IL-21 stimulated B cells (n=8) were determined and shown as mean % of no Vitamin D control (right panel). B. IgG and IgM production determined by ELISA. 31 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , The results are the mean?SEM and are representative of five independent experiments. Significant differences are shown as ** (p<0.01) or *** (p<0.001). C. IgG (left) IgM (right) secreting cells were determined at day 7 by ELISPOT. Data are the mean?SEM from triplicate determinations and are representative of one of three independent experiments. D. Purified B cells were cultured with anti-CD40 and IL21 for 5 days, after which 1,25(OH) D (10 nM) was 2 3 added to the culture. The effects of Vitamin D on persistence of plasma cells and Ig production over the next 5 days were assessed and were compared to a no Vitamin D controls. Data are the mean ?SEM from triplicate determinates. Significant differences are shown as * (p<0.05). 6. 1,25(OH)2D3能抑制未活化 B细胞向类转变后的记忆细胞的分化 为了解 1,25(OH)2D3的抑制效应是否只针对浆细胞的产生,我们又探讨了其在 调节类转变后的记忆 B细胞形成过程中的可能作用。首先我们通过细胞分选得到纯 度高于 99%的未活化 B细胞亚群( naïve B cells, (CD19+, IgG-CD27-)),见图 6A 。并 应用已知可以诱导类转变过程的条件进行体外培养 (26),如图 6B所示, IL21在与 anti-CD40或 anti-IgM/ anti-CD40共刺激条件下,B细胞在第 5天可发生类转变表达 表面 IgG分子。值得注意的是,1,25(OH)2D3 处理的 B细胞中,表面 IgG阳性的 B 细胞比例显著减少 (图 6B,C),而表面 IgM阳性的比例却明显增加 (图 6B)。与此结 果相一致的是,在其后第 7天浆细胞的分化以及 IgG的产生中,1,25(OH)2D3同样 显示其抑制作用(图 6D)。由此证实, 1,25(OH)2D3 不仅能抑制浆细胞的产生还能 抑制类转变后的记忆细胞的产生。 32 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 图 6. 1,25(OH)2D3对 B细胞类转变过程的影响 Figure 6. Vitamin D inhibits generation of post-switch memory B cells from naïve B cells. A. Pre-switched naïve B cells (CD19+IgG-CD27-) were sorted from enriched peripheral B cells from three healthy donors. Post-sort analysis of the naïve B cells is shown. The purity of sorted cells was more than 99%. B. Naïve B cells were stimulated as indicated with or without 1,25(OH) D (10 nM) . After 5 days in culture, the B cells were stained for expression 2 3 33 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , of CD19 and IgG or IgM (upper right quadrant). C. The percentages of post-switched memory B cells were compared between culture with or without Vitamin D. D. After 7 days in culture, the number of plasma cells and IgG in the supernatants were analyzed. Data are the mean ? SEM from triplicate determinations of one of three independent experiments. Asterisks indicate the statistical significance between Vitamin D treated and control B cells (p?0.01). 7. 1,25(OH)2D3抑制 XBP1和 ERN1在激活 B细胞上的表达 至此,我们已经证实 1,25(OH)2D3 能减少 B细胞增生、类转变后的记忆 B细 胞以及浆细胞的产生,以及 Ig的分泌。然而,1,25(OH)2D3究竟在细胞分化的哪个 阶段发挥其作用尚不得而知。为此,我们检测了在 B细胞成熟和分化过程中起关键 作用的转录因子的表达,如与生发中心反应相关的转录因子 PAX5、 BCL6、和 AID;以及浆细胞分化相关的转录因子 BLIMP1, IRF4, MTA3, XBP1和 ERN1 (27)。 所有的检测均在培养第 3天进行,因为这时 1,25(OH)2D3影响记忆细胞和浆细胞比 例的作用尚未显现。结果显示, 1,25(OH)2D3对 B细胞表达 PAX5, BCL6, AID, BLIMP1, MAT3或 IRF4没有影响。然而,即使是在这个相对较早的观察点,XBP1 和 ERN1的表达已经有了显著的下降 (图 7)。尽管如此,由于 1,25(OH)2D3对调节 记忆细胞和浆细胞分化这一程序缺乏一致性结果,我们相信 1,25(OH)2D3可能是通 过另外的途径对 B细胞产生抑制作用。 34 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 图 7. 1,25(OH)2D3对浆细胞分化相关的转录因子表达的影响 A. 2 0 0 0 1 0 8 1 6 0 0 nil VitD 1 2 0 0 6 8 0 0 4 4 0 0 2 0 0 AID Pax5 Bcl6 N=3 N=3 N=3 B. 15 nil 12 VitD 9 6 * 3 * 0 Blimp1 IRF4 MAT3 XBP1 ERN1 N=5 N=4 N=4 N=3 N=3 Figure 7. Vitamin D inhibits XBP1 and ERN1 expression by activated B cells. RNAs were extracted from freshly isolated B cells and B cells cultured with 2 3 anti-IgM, anti-CD40 and IL-21 with or without 1,25(OH) D (10 nM) for 3 days. The mRNA expression of various gene products was determined by RT-PCR and the fold change was compared to freshly isolated B cells. A. Relative expression of PAX, BCL6, and AID. B. Relative expression of BLIMP1, IRF4, MAT3, XBP1 and ERN1. Data represent the mean ? SEM of three independent experiments conducted in triplicate from different donors. Significant differences between Vitamin D and control responses are shown by * (p<0.05). 8. 1,25(OH)2D3对NF-κB调节基因表达的影响 为进一步明确1,25(OH)2D3对B细胞作用的机制,首先我们对 NF-κB介导的信号 传导途径相关的一系列基因进行筛选。如表1所示,虽然有个别基因的mRNA表达水 平在1,25(OH)2D3作用下出现改变,但纵观整体,并没有发现 NF-κB信号途径的主要 基因有一致性的变化。 35 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 表1.维生素D对NF-κB调节基因表达的影响 mRNA Mean of fold change P value 0.55 0.09 Angiotensinogen (serpin peptidase inhibitor, clade A, member 8) 1.10 V-akt murine thymoma viral oncogene homolog 1 0.34 Activating transcription factor 1 1.19 0.11 B-cell CLL/lymphoma 10 1.16 0.11 B-cell CLL/lymphoma 3 2.12 0.16 B-factor, properdin 0.68 0.05 Baculoviral IAP repeat-containing 2 0.73 0.12 Caspase recruitment domain family, member 4 0.79 0.07 Caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase) 0.85 0.29 Caspase 8, apoptosis-related cysteine peptidase 0.87 0.35 Chemokine (C-C motif) ligand 2 0.09 0.12 CD40 antigen (TNF receptor superfamily member 5) 0.85 0.20 CASP8 and FADD-like apoptosis regulator 1.29 0.12 Conserved helix-loop-helix ubiquitous kinase 1.08 0.42 Colony stimulating factor 2 (granulocyte-macrophage) 0.51 0.27 Colony stimulating factor 3 (granulocyte) 0.63 0.16 Solute carrier family 44, member 2 1.45 0.00 EDAR-associated death domain 0.48 0.04 Endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2 0.89 0.39 Early growth response 1 1.37 0.22 ELK1, member of ETS oncogene family 0.88 0.30 Coagulation factor II (thrombin) receptor 0.59 0.10 Fas (TNFRSF6)-associated via death domain 0.88 0.25 Fas ligand (TNF superfamily, member 6) 0.84 0.37 0.83 V-fos FBJ murine osteosarcoma viral oncogene homolog 0.32 Gap junction protein, alpha 1, 43kDa (connexin 43) 0.16 0.20 Heme oxygenase (decycling) 1 1.36 0.21 5-hydroxytryptamine (serotonin) receptor 2B 0.64 0.04 0.91 Intercellular adhesion molecule 1 (CD54), human rhinovirus receptor 0.40 Interferon, alpha 1 0.61 0.16 Interferon, beta 1, fibroblast 0.55 0.14 Interferon, gamma 0.80 0.39 1.11 Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta 0.39 Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon 0.86 0.33 Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma 0.81 0.04 Interleukin 10 0.81 0.27 Interleukin 1, alpha 0.70 0.01 Interleukin 1, beta 0.38 0.05 Interleukin 1 receptor, type I 0.71 0.14 Interleukin 6 (interferon, beta 2) 3.27 0.01 Interleukin 8 0.76 0.22 Interleukin-1 receptor-associated kinase 1 0.66 0.19 Interleukin-1 receptor-associated kinase 2 3.09 0.09 V-jun sarcoma virus 17 oncogene homolog (avian) 0.93 0.36 0.60 Lymphotoxin alpha (TNF superfamily, member 1) 0.15 Lymphotoxin beta receptor (TNFR superfamily, member 3) 0.96 0.47 Mucosa associated lymphoid tissue lymphoma translocation gene 1 0.88 0.24 Mitogen-activated protein kinase kinase kinase 1 1.25 0.23 1.01 0.48 Myeloid differentiation primary response gene (88) 0.26 NACHT, leucine rich repeat and PYD containing 12 0.19 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) 0.95 0.40 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) 1.00 0.50 1.56 0.03 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha 0.91 Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform 0.28 V-raf-1 murine leukemia viral oncogene homolog 1 0.92 0.38 V-rel reticuloendotheliosis viral oncogene homolog (avian) 0.70 0.21 V-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gen 1.10 0.36 0.84 V-rel reticuloendotheliosis viral oncogene homolog B, nuclear factor of kappa light polypeptide gen 0.23 Ret finger protein 2 0.92 0.24 Ras homolog gene family, member A 1.08 0.36 Receptor (TNFRSF)-interacting serine-threonine kinase 1 1.19 0.25 0.80 Solute carrier family 20 (phosphate transporter), member 1 0.20 Signal transducer and activator of transcription 1, 91kDa 1.01 0.49 TANK-binding kinase 1 0.98 0.45 0.55 0.02 Toll-like receptor adaptor molecule 2 0.75 Toll-like receptor 1 0.16 Toll-like receptor 2 0.30 0.10 Toll-like receptor 3 1.47 0.26 1.04 0.46 Toll-like receptor 4 0.83 Toll-like receptor 6 0.07 Toll-like receptor 7 0.65 0.01 Toll-like receptor 8 0.72 0.34 0.69 0.22 Toll-like receptor 9 Transmembrane emp24 protein transport domain containing 4 0.99 0.48 0.48 Tumor necrosis factor (TNF superfamily, member 2) 0.07 Tumor necrosis factor, alpha-induced protein 3 0.74 0.24 Tumor necrosis factor receptor superfamily, member 10a 0.56 0.05 0.82 0.24 Tumor necrosis factor receptor superfamily, member 10b 0.53 Tumor necrosis factor receptor superfamily, member 1A 0.01 Tumor necrosis factor receptor superfamily, member 7 0.95 0.44 Tumor necrosis factor (ligand) superfamily, member 10 1.16 0.28 1.82 0.23 Tumor necrosis factor (ligand) superfamily, member 14 1.28 TNFRSF1A-associated via death domain 0.10 Toll-like receptor adaptor molecule 1 1.39 0.10 36 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 9. 1,25(OH)2D3对细胞周期相关调节基因表达的影响 接着,我们应用人细胞周期相关基因 PCR Array检测了1,25(OH)2D3 对一系列 细胞周期调节基因的影响 (见表2)。结果发现,在1,25(OH)2D3作用下,有相当一部 分基因包括细胞周期蛋白 cyclin D1、cyclin D2, cyclin T1, cyclin T2,细胞周期蛋白依 赖性激酶(cyclin-dependent kinase,CDK) 4和 6在激活的B细胞中的表达都显 著性下降。与其相对应的, CDK抑制剂 p27的表达则明显上调 ,而其他相关基因包 括CDK抑制剂p21无变化。通过常规的 RT-PCR,我们得到了同样的结果,即 1,25(OH)2D3使激活的B细胞p27的mRNA表达明显上调,而 p21或p18无变化(图8)。 图8. 1,25(OH)2D3对细胞周期相关调节基因表达的影响 A. 60000 nil * VitD 45000 30000 15000 0 p21 p27 B. 80000 nil VitD 60000 * 40000 20000 0 p21 p27 p18 Figure 8. The effect of Vitamin D on cell cycle regulated genes. RNAs were extracted from freshly isolated B cells and B cells cultured with anti-IgM, 2 3 anti-CD40 and IL-21 with or without 1,25(OH) D (10nM) for 3 days. The mRNA expression of P27, P21 and P18 was determined by superarray and/or quantitative PCR. A. The relative expression of P21 and P27 determined by Superarray. Data represent the mean? SEM of three independent experiments. B. The relative expression of P21, P27 and P18 determined by quantitative PCR. Data represent the mean? SEM of three independent experiments. Significant 37 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , differences between Vitamin D treated and control B cells are shown by * (p<0.05). 表2. 1,25(OH)2D3对细胞周期相关调节基因表达的影响 mRNA Mean of fold change P value V-abl Abelson murine leukemia viral oncogene homolog 1 0.55 0.07 Anaphase promoting complex subunit 2 0.86 0.22 Anaphase promoting complex subunit 4 0.76 0.19 DIRAS family, GTP-binding RAS-like 3 1.67 0.26 Ataxia telangiectasia mutated (includes complementation groups A, C and D) 1.03 0.31 Ataxia telangiectasia and Rad3 related 0.95 0.30 BCL2-associated X protein 1.32 0.50 BRCA2 and CDKN1A interacting protein 0.72 0.03 B-cell CLL/lymphoma 2 1.13 0.41 Baculoviral IAP repeat-containing 5 (survivin) 1.11 0.47 Breast cancer 1, early onset 0.83 0.12 Breast cancer 2, early onset 0.86 0.23 Cyclin B1 0.83 0.13 Cyclin B2 0.85 0.26 Cyclin C 1.03 0.38 Cyclin D1 0.40 0.14 Cyclin D2 0.72 0.03 Cyclin E1 0.86 0.04 Cyclin F 1.03 0.39 Cyclin G1 1.62 0.20 Cyclin G2 0.99 0.45 Cyclin H 1.29 0.44 Cyclin T1 0.83 0.04 Cyclin T2 1.15 0.03 CDC16 cell division cycle 16 homolog (S. cerevisiae) 0.75 0.15 Cell division cycle 2, G1 to S and G2 to M 1.08 0.46 CDC20 cell division cycle 20 homolog (S. cerevisiae) 0.97 0.24 Cell division cycle 34 1.05 0.45 Cyclin-dependent kinase 2 1.07 0.33 Cyclin-dependent kinase 4 0.82 0.04 Cyclin-dependent kinase 5, regulatory subunit 1 (p35) 0.68 0.07 CDK5 regulatory subunit associated protein 1 0.70 0.01 Cyclin-dependent kinase 6 0.83 0.12 Cyclin-dependent kinase 7 (MO15 homolog, Xenopus laevis, cdk-activating kinase) 0.99 0.43 Cyclin-dependent kinase 8 0.79 0.13 Cyclin-dependent kinase inhibitor 1A (p21, Cip1) 0.74 0.08 Cyclin-dependent kinase inhibitor 1B (p27, Kip1) 2.44 0.03 Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) 1.65 0.38 Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) 0.60 0.11 Cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase) 0.96 0.35 CHK1 checkpoint homolog (S. pombe) 0.72 0.19 CHK2 checkpoint homolog (S. pombe) 0.83 0.17 CDC28 protein kinase regulatory subunit 1B 0.76 0.08 CDC28 protein kinase regulatory subunit 2 0.86 0.04 Cullin 1 0.98 0.36 Cullin 2 0.74 0.14 Cullin 3 0.71 0.10 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 (CHL1-like helicase homolog, S. cerevisiae) 1.13 0.23 Dynamin 2 1.13 0.31 E2F transcription factor 4, p107/p130-binding 0.82 0.23 Growth arrest and DNA-damage-inducible, alpha 1.85 0.22 General transcription factor IIH, polypeptide 1, 62kDa 0.96 0.26 G-2 and S-phase expressed 1 0.93 0.15 Hect domain and RLD 5 0.84 0.13 HUS1 checkpoint homolog (S. pombe) 0.85 0.18 Kinetochore associated 1 0.79 0.01 Karyopherin alpha 2 (RAG cohort 1, importin alpha 1) 0.74 0.05 MAD2 mitotic arrest deficient-like 1 (yeast) 0.92 0.27 MAD2 mitotic arrest deficient-like 2 (yeast) 0.81 0.05 MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae) 0.90 0.14 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) 1.19 0.42 MCM4 minichromosome maintenance deficient 4 (S. cerevisiae) 0.79 0.13 MCM5 minichromosome maintenance deficient 5, cell division cycle 46 (S. cerevisiae) 1.21 0.32 Antigen identified by monoclonal antibody Ki-67 0.65 0.04 Menage a trois 1 (CAK assembly factor) 0.72 0.05 MRE11 meiotic recombination 11 homolog A (S. cerevisiae) 0.73 0.02 Nibrin 0.87 0.14 Proliferating cell nuclear antigen 0.83 0.09 RAD1 homolog (S. pombe) 1.01 0.48 RAD17 homolog (S. pombe) 1.07 0.12 RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae) 1.56 0.22 RAD9 homolog A (S. pombe) 0.96 0.44 Retinoblastoma 1 (including osteosarcoma) 0.94 0.27 Retinoblastoma binding protein 8 0.85 0.14 Retinoblastoma-like 1 (p107) 1.31 0.27 Retinoblastoma-like 2 (p130) 0.97 0.37 Replication protein A3, 14kDa 1.36 0.22 SERTA domain containing 1 1.35 0.18 S-phase kinase-associated protein 2 (p45) 0.70 0.18 SMT3 suppressor of mif two 3 homolog 1 (yeast) 0.72 0.02 Transcription factor Dp-1 0.85 0.11 Transcription factor Dp-2 (E2F dimerization partner 2) 0.91 0.26 Tumor protein p53 (Li-Fraumeni syndrome) 0.97 0.37 Ubiquitin-activating enzyme E1 (A1S9T and BN75 temperature sensitivity complementing) 2.86 0.21 38 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 10( B淋巴细胞能表达维生素 D羟化酶CYP27B1,使25(OH)D3活化成1,25(OH)2D3. 已知血浆中存在的 25(OH)D3,要在1α-羟化酶 (CYP27B1)的作用下最后转变成 具有生物活性的1,25(OH)2D3,而后者主要由肾脏组织产生,也有报道单核细胞和巨 噬细胞也能局部性表达该酶 (3, 4)。应用RT-PCR方法,我们检测了 1α-羟化酶在人 外周B细胞上的表达。如图9A所示, B细胞能构成性地表达1α-羟化酶的mRNA,在 激活作用下其表达水平明显增高,但 1,25(OH)2D3. 对其影响不明显。为确认该酶的 表达上调能相应地产生功能性结果,我们用 anti-CD40和IL-21激活纯化的B细胞,并 在培养液中分别加入1,25(OH)2D3.(10 nM)或不同浓度的25(OH)D3,观察CD38表达、 浆细胞分化以及 IgG产生在各组间的差异 (图9B)。结果发现1,25(OH)2D3的前体, 25(OH)D3,在25倍以上的浓度条件下,可以对 B细胞产生与活性成分相似的作用。 考虑到单核细胞的污染可能误导这一结果,我们分选了人扁桃腺的 B细胞进行了类 似的实验,得到了同样的结果。由此,我们推测 B细胞自身能产生 1α-羟化酶,使 25(OH)D3 转化成有活性的1,25(OH)2D3,并继而抑制B细胞功能 图9. B细胞能使25(OH)D3活化成1,25(OH)2D3 Figure 9. B cells can convert inactive 25(OH)D3 into the active form of Vitamin D. A. CYP27B1 is constitutively expressed by fresh B cells and is induced by stimulation. B cells were analyzed either immediately after isolation or were 39 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , incubated for 3 days with the indicated stimuli with or without 1,25(OH)2D3 (10nM). The expression of CYP27B1 was determined by RT-PCR relative to B2M mRNA (×106). The means ? SEM of one of three independent experiments are shown . Significant differences between Vitamin D treated and control B cells are shown by * (p<0.05). Significant differences between day 0 and stimulated B cells are shown as ** (p<0.01) B. Purified B cells were stimulated with anti-CD40 and IL-21 with either 10 nM 1,25(OH)2D3 or different concentrations of 25(OH)D3 ranging from 0 to 1000 nM. After 7 days in culture, the cells were washed and stained for expression of CD19, CD27 and CD38. The geometric mean fluorescence of CD38 and the frequency of plasma cells (CD27hi/CD38hi) were determined. The culture supernatants were analyzed for IgG production, The 25(OH)D3 dose response (solid line) is compared to 10 nM 1,25(OH)2D3 (dashed line) 讨 论 本研究中,我们证实了1,25(OH)2D3 人外周B淋巴细胞具有直接有效的免疫调节 作用,包括抑制B细胞增生、减少类转变后记忆B细胞和浆细胞的分化、以及 Ig的 产生。 1,25-二羟维生素D3 (1,25(OH)2D3),是维生素D3的生物活性成分,不仅调节骨骼 和钙磷代谢,还在免疫系统中发挥重要作用。这些免疫调节效应被认为是通过其对 抗原递呈细胞(主要是DC)和T细胞的作用完成的 (7-10)。以往人们广泛接受的观 点是其对B细胞并没有直接作用,而是通过抑制 CD4+ T细胞功能或抑制单核细胞或 巨噬细胞产生的细胞因子而间接完成的 (12, 28)。然而,我们的研究则明确证明了 1,25(OH)2D3对B细胞功能有直接抑制作用。重要的一点是,本研究中对 B细胞起作 用的1,25(OH)2D3浓度完全在人正常血浆浓度范围内 (29, 30)。 1,25(OH)2D3的主要生物功能是通过与 VDR结合而发挥的。以往关于VDR在B细 胞中的表达一直有争议。有学者研究了 VDR在淋巴细胞的表达,认为CD8+T细胞最 高,其次为CD4+T细胞,而B细胞不表达(13)。另一项研究则报道人扁桃腺中的 B细 胞能表达VDR,并在激活状态下上调表达水平 (24)。也有学者发现B细胞淋巴瘤细 胞株SUDHL4和SUDHL5同样有VDR的表达 (31)。本研究中,我们证实人外周 B细 胞能结构性地低水平表达 VDR mRNA,在激活状态下其表达水平相应升高,而 1,25(OH)2D3 则能进一步提高其表达,这一作用呈时间依赖效应。这些结果提示 40 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 1,25(OH)2D3对于不同激活程度的B细胞可能有着不同的作用,而对于血浆维生素 D3 水平不同的个体, 1,25(OH)2D3的生物作用也会有差异。 1,25(OH)2D3主要通过24-羟化酶(CYP24A1)降解失活 (2)。应用基因芯片技术 对人结肠癌和卵巢肿瘤细胞株的分析发现, CYP24A1是1,25(OH)2D3最明显的诱导 基因(32, 33)。本研究中,我们同样证实了在 1,25(OH)2D3作用下,激活的 B细胞显 著上调CYP24A1的表达。与VDR不同的是, CYP24A1的表达并不受激活程度的影 响。这些结果再次证实, 1,25(OH)2D3可以直接作用于人外周 B细胞,同时也提示维 生素D对B细胞的作用不仅受 VDR表达水平的影响,还受到细胞降解该活性分子能 力的影响。而激活的B细胞对1,25(OH)2D3反应能力的提高则仅归因于细胞 VDR表达 水平的提高,与CYP24A1的表达无关。 25(OH)D3-1-α-羟化酶 (CYP27B1),可以使25(OH)D3完成最后的羟化作用形成 活性1,25(OH)2D3,这一过程主要在肾脏近曲小管表达 (2)。有趣的事,我们的结果显 示静止的B细胞同样能表达CYP27B1 mRNA,体外激活后其表达水平显著性上升, 而1,25(OH)2D3对其影响不明显。更重要的事,我们发现1,25(OH)2D3的前体成分, 25(OH)D3,在高浓度下同样对纯化的 B细胞有生物效应。由此推测,B细胞本身能使 25(OH)D3代谢形成1,25(OH)2D3,从而象巨噬细胞和树突状细胞一样成为另一个肾 外合成的 1,25(OH)2D3 来源 (3, 4)。而在SLE这样的疾病中,由于存在广泛的B细胞 过度活化 (20),其对维生素D代谢的影响可能就显得更为重要。 1,25(OH)2D3 抑制淋巴细胞和多种人肿瘤细胞株增生的能力多有报道 (35)。本 研究证实1,25(OH)2D3同样对B细胞增生有抑制作用。通过 CFSE荧光标记法分析 , 1,25(OH)2D3对B细胞增生的抑制作用并非针对最初的分裂,而是其后的增生过程。 这一结果与 B细胞激活和 1,25(OH)2D3介导的 VDR表达上调的结果相呼应,提示 1,25(OH)2D3的抗增生作用是以一定量的 VDR表达为前提。而且,这一作用还同时与 活化B细胞的凋亡有关。 1,25(OH)2D3 抑制B细胞增生和诱导凋亡的作用很可能直接导致了我们所观察 到的其对浆细胞分化和 Ig产生的抑制作用。有观点认为,浆细胞分化需要最初的 B 细胞增生 (36)以及一定的分裂次数 (37)。以往的研究证实,用IL-21和 anti-CD40, 或同时联合BCR-交联可以有效地体外诱导 B细胞增生和浆细胞分化,而所有浆细胞 41 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 都是经过增生后产生的 (26)。应用这一体系,我们证实 1,25(OH)2D3对浆细胞分化 和Ig产生有抑制作用。有趣的是 ,当1,25(OH)2D3在B细胞体外刺激后的第 5天加入 时,这一抑制作用就不复存在,提示 1,25(OH)2D3能抑制浆细胞产生,而对已经产 生的浆细胞则没有作用。 B细胞在成熟和浆细胞分化的不同阶段需要多个转录因子参与,这些转录因子 之间互相调节构成网络 (38)。本研究的结果显示 1,25(OH)2D3对这一网络中绝大多数 转录因子的表达并无影响,包括 PAX5, BCL6, AID, BLIMP1, MTA3和IRF4。因此, 尚没有充分的证据证明 1,25(OH)2D3是通过直接作用于这些转录因子而抑制记忆 B细 胞或浆细胞分化的。尽管如此,我们的结果也显示XBP1和ERN1(XBP1 mRNA产 生剪切异构体编码更为稳定、活性更强蛋白过程所需的转录因子)的表达在 1,25(OH)2D3作用下显著性下调。由于 XBP1是调节B细胞分化过程最下游的转录因 子,且直接与浆细胞产生、Ig产生相关,这一结果至少可以部分解释1,25(OH)2D3 对 Ig产生以及对Ig分泌型B细胞的抑制作用要大于对表型定义的浆细胞产生的抑制作 用。换言之,1,25(OH)2D3下调B细胞表达XBP1和ERN1 mRNA,可以减少每个表型 定义的浆细胞产生的Ig量。然而,这一结果并不能解释1,25(OH)2D3 对记忆B细胞形 成以及B细胞增生的抑制作用。 近年来,不断有研究对1,25(OH)2D3参与免疫调节作用的分子学基础进行探索。 以往研究表明 1,25(OH)2D3对数个重要的细胞内信号传导通路有作用,包括 NF-κB 和细胞周期进程。有报道 1,25(OH)2D3对T细胞、单核细胞或巨噬细胞的作用是通 过抑制NF-κB信号传导途径发挥的 (9, 40,41)。然而,在本研究中我们应用人 NF-κB 信号传导途径 PCR-Array对84个相关基因在 B细胞中的表达进行筛选 ,结果发现 1,25(OH)2D3对这一途径中的关键基因并无整体的抑制作用。细胞周期指一个细胞 经生长、分裂而增殖成两个所经历的全过程,通常可分为若干阶段,即 G1期、S期、 G2期和M期,该过程受一系列正负信号的调节。真核细胞中细胞周期蛋白 (cyclin)和 cyclin依赖性激酶(CDKs)形成的二聚体复合物为细胞周期调控的核心机制,后者又 42 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 受到CDK抑制剂的负调节。由于 1,25(OH)2D3直接抑制活化B细胞的增生,我们检 , 测了1,25(OH)2D3是否对B细胞周期调节蛋白的表达有抑制作用,结果发现 CDK4、 CDK6以及cyclin D mRNA表达水平下调,而CDK抑制剂p27表达上调。即往已有报 道,1,25(OH)2D3可以通过诱导不同的 CDK抑制剂(p21和/或p27)的基因转录, 使细胞由G1期向S期转化的过程受阻,其针对的 CDK抑制剂因细胞种类不同而不同 (42 -44)。肿瘤研究中,免疫组化结果证实小鼠体内注射 1,25(OH)2D3 能有效地提高 肿瘤细胞中p27的表达,并缩小肿瘤体积 (45)。并有多个研究探讨了其相关的机理, 其中包括1)VDR-Sp1与p27启动子相互作用介导的 p27转录增加(46);2)VDR诱导 的CDK2活性降低以及Skp2含量减少(其为p27降解的主要影响因素),使p27蛋白稳 定性增加;3)诱导p27去磷酸化酶PTEN的产生 (44)。CDK抑制剂在B细胞反应中起 重要作用,比如p18就被认为是B细胞终止增生并最终分化成功能性的浆细胞所必需 的(42, 47, 48)。还有研究证实p18和p27在调节B细胞增生的不同阶段发挥作用,前者 主要通过阻断CDK4、CDK6和cyclin D的相互作用使B细胞停留在G1期,而p27则 能与多种CDK和cyclin作用,并影响整个细胞周期进程的进入(48)。我们的研究结果 则显示, 1,25(OH)2D3能上调人激活状态下外周 B细胞的p27 mRNA表达,而对p21 或p18无作用,提示1,25(OH)2D3可能通过上调p27的表达,阻止周期中的 B细胞进入 下一个周期,并最终对 B细胞增生产生抑制作用。由此, 1,25(OH)2D3 介导的p27表 达上调可能在B细胞对1,25(OH)2D3的反应中起重要作用。这也再次提示1,25(OH)2D3 对于浆细胞和记忆 B细胞分化的抑制作用可能还应主要归功于其对分化过程开始之 前对B细胞增生的抑制作用 (36)。 1,25(OH)2D3除了能抑制浆细胞分化和 Ig产生,我们的结果还证实它对记忆 B细 43 上海交通大学,, 届博士论文,,, ,,,,,,,,,,,,,,,, , , , , , , , , , , , , , , , , , , 胞的分化同样有抑制作用。记忆细胞和浆细胞比例增高是狼疮患者 B细胞异常的两 大特征(54)。与未活化B细胞和浆细胞不同,各种传统的免疫抑制治疗通常不针对记 忆B细胞,这就给疾病的复发带来隐患 (55)。维生素D因为能同时阻止或减少记忆 B 细胞产生而为诱导SLE缓解提供了新的治疗手段。事实上, 1,25(OH)2D3治疗狼疮在 小鼠试验已经初见成效,自发性狼疮鼠模型MRL/lpr注射1,25(OH)2D3或其类似物后 可以明显减少蛋白尿,延长存活期 (56)。 结论:1,25(OH)2D3对维持B细胞免疫稳态有重要作用,纠正维生素 D缺乏不仅可以 防治骨质疏松症,还有助于抑制自身免疫性疾病SLE中的B细胞过度活化,是一种新 的有效的免疫治疗手段。 44 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, ,,,参考文献 ,,,,,,,, 参考文献 1. van Etten, E., and Mathieu, C. 2005. Immunoregulation by 1,25-dihydroxyvitamin D-3: Basic concepts. Journal of Steroid Biochemistry and Molecular Biology 97:93-101. 2. Jones, G., Strugnell, S.A., and DeLuca, H.F. 1998. Current understanding of the molecular actions of vitamin D. Physiological Reviews 78:1193-1231. 3. Overbergh, L., Decallonne, B., Valckx, D., Verstuyf, A., Depovere, J., Laureys, J., Rutgeerts, O., Saint-Arnaud, R., Bouillon, R., and Mathieu, C. 2000. Identification and immune regulation of 25-hydroxyvitamin D-1-alpha-hydroxylase in murine macrophages. Clinical and Experimental Immunology 120:139-146. 4. Hewison, M., Freeman, L., Hughes, S.V., Evans, K.N., Bland, R., Eliopoulos, A.G., Kilby, M.D., Moss, P.A.H., and Chakraverty, R. 2003. Differential regulation of vitamin D receptor and its ligand in human monocyte-derived dendritic cells. Journal of Immunology 170:5382-5390. 5. Haussler, M.R., Whitfield, G.K., Haussler, C.A., Hsieh, J.C., Thompson, P.D., Selznick, S.H., Dominguez, C.E., and Jurutka, P.W. 1998. The nuclear vitamin D receptor: Biological and molecular regulatory properties revealed. Journal of Bone and Mineral Research 13:325-349. 6. Mathieu, C., and Adorini, L. 2002. The coming of age of 1,25-dihydroxyvitamin D-3 analogs as immunomodulatory agents. Trends in Molecular Medicine 8:174-179. 7. Penna, G., and Adorini, L. 2000. 1 alpha,25-dihydroxyvitamin D-3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. Journal of Immunology 164:2405-2411. 8. van Halteren, A.G.S., van Etten, E., de Jong, E.C., Bouillon, R., Roep, B.O., and Mathieu, C. 2002. Redirection of human autoreactive T-cells upon interaction with dendritic cells modulated by TX527, an analog of 1,25 dihydroxyvitamin D-3. Diabetes 51:2119-2125. 9. D'Ambrosio, D., Cippitelli, M., Cocciolo, M.G., Mazzeo, D., Di Lucia, P., Lang, R., Sinigaglia, F., and Panina-Bordignon, P. 1998. Inhibition of IL-12 production by 1,25-dihydroxyvitamin D-3 - Involvement of NF-kappa B downregulation in transcriptional repression of the p40 gene. Journal of Clinical Investigation 101:252-262. 10. Boonstra, A., Barrat, F.J., Crain, C., Heath, V.L., Savelkoul, H.F.J., and O'Garra, A. 2001. 1 alpha,25-dihydroxyvitamin D3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells. Journal of Immunology 167:4974-4980. 11. Cippitelli, M., Fionda, C., Di Bona, D., Di Rosa, F., Lupo, A., Piccoli, M., Frati, L., and Santoni, A. 2002. Negative regulation of CD95 ligand gene expression by vitamin D3 in T lymphocytes. Journal of Immunology 168:1154-1166. 12. Muller, K., Heilmann, C., Poulsen, L.K., Barington, T., and Bendtzen, K. 1991. The Role of Monocytes and T-Cells in 1,25-Dihydroxyvitamin-D3 Mediated Inhibition of B-Cell Function-Invitro. Immunopharmacology 21:121-128. 13. Veldman, C.M., Cantorna, M.T., and DeLuca, H.F. 2000. Expression of 45 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, ,,,参考文献 ,,,,,,,, 1,25-dihydroxyvitamin D-3 receptor in the immune system. Archives of Biochemistry and Biophysics 374:334-338. 14. Huisman, A.M., White, K.P., Algra, A., Harth, M., Vieth, R., Jacobs, J.W.G., Bijlsma, J.W.J., and Bell, D.A. 2001. Vitamin D levels in women with systemic lupus erythematosus and fibromyalgia. Journal of Rheumatology 28:2535-2539. 15. Becker, A., Fischer, R., and Schneider, M. 2001. Bone density and 25-OH-vitamin D in systemic lupus erythematosus. Zeitschrift Fur Rheumatologie 60:352-358. 16. Gladman, D.D., Urowitz, M.B., Esdaile, J.M., Hahn, B.H., Klippel, J., Lahita, R., Liang, M.H., Schur, P., Petri, M., and Wallace, D. 1999. Guidelines for referral and management of systemic lupus erythematosus in adults. Arthritis and Rheumatism 42:1785-1796. 17. Anolik, J.H., and Aringer, M. 2005. New treatments for SLE: cell-depleting and anti-cytokine therapies. Best Practice & Research in Clinical Rheumatology 19:859-878. 18. Looney, R.J., Anolik, J.H., Campbell, D., Felgar, R.E., Young, F., Arend, L.J., Sloand, J.A., Rosenblatt, J., and Sanz, I. 2004. B cell depletion as a novel treatment for systemic lupus erythematosus - A phase I/II dose-escalation trial of rituximab. Arthritis and Rheumatism 50:2580-2589. 19. Van den Bergh, B., Selleslag, D., Boelaert, J.R., Matthys, E.G., Schurgers, M., Vandecasteele, S., and De Vriese, A. 2005. Management of therapy-resistant systemic lupus erythematosus with rituximab: Report of a case and review of the literature. Acta Clinica Belgica 60:102-105. 20. Grammer, A.C., and Lipsky, P.E. 2003. B cell abnormalities in systemic lupus erythematosus. Arthritis Research & Therapy 5:S22-S27. 21. Hochberg, M.C. 1997. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis and Rheumatism 40:1725-1725. 22. Bombardier, C., Gladman, D.D., Urowitz, M.B., Caron, D., and Chang, C.H. 1992. Derivation of the Sledai - a Disease-Activity Index for Lupus Patients. Arthritis and Rheumatism 35:630-640. 23. Stoeckler, J.D., Stoeckler, H.A., Kouttab, N., and Maizel, A.L. 1996. 1 alpha,25-dikydroxyvitamin D-3 modulates CD38 expression on human lymphocytes. Journal of Immunology 157:4908-4917. 24. Morgan, J.W., Kouttab, N., Ford, D., and Maizel, A.L. 2000. Vitamin D-Mediated gene regulation in phenotypically defined human B cell subpopulations. Endocrinology 141:3225-3234. 25. Joshua, D., Pope, B., Brown, R., Barrow, L., Murray, A., and Gibson, J. 2002. Phenotyping primitive plasma cells. British Journal of Haematology 117:252-253. 26. Ettinger, R., Sims, G.P., Fairhurst, A.M., Robbins, R., da Silva, Y.S., Spolski, R., Leonard, W.J., and Lipsky, P.E. 2005. IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. Journal of Immunology 175:7867-7879. 27. Matthias, P., and Rolink, A.G. 2005. Transcriptional networks in developing and mature B cells. Nature Reviews Immunology 5:497-508. 28. Chen, W.C., Vayuvegula, B., and Gupta, S. 1987. 46 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, ,,,参考文献 ,,,,,,,, 1,25-Dihydroxyvitamin-D3-Mediated Inhibition of Human B-Cell Differentiation. Clinical and Experimental Immunology 69:639-646. 29. Harris, S.S. 2006. Vitamin D and African Americans. Journal of Nutrition 136:1126-1129. 30. Grant, W.B. 2006. Epidemiology of disease risks in relation to vitamin D insufficiency. Progress in Biophysics & Molecular Biology 92:65-79. 31. Hickish, T., Cunningham, D., Colston, K., Millar, B.C., Sandle, J., Mackay, A.G., Soukop, M., and Sloane, J. 1993. The Effect of 1,25-Dihydroxyvitamin-D(3) on Lymphoma Cell-Lines and Expression of Vitamin-D Receptor in Lymphoma. British Journal of Cancer 68:668-672. 32. Zhang, X.H., Li, P.F., Bao, J.Y., Nicosia, S.V., Wang, H.G., Enkemann, S.A., and Bai, W.L. 2005. Suppression of death receptor-mediated apoptosis by 1,25-dihydroxyvitamin D-3 revealed by microarray analysis. Journal of Biological Chemistry 280:35458-35468. 33. Wood, R.J., Tchack, L., Angelo, G., Pratt, R.E., and Sonna, L.A. 2004. DNA microarray analysis of vitamin D-induced gene expression in a human colon carcinoma cell line. Physiological Genomics 17:122-129. 34. Linker-Israeli, M., Elstner, E., Klinenberg, J.R., Wallace, D.J., and Koeffler, H.P. 2001. Vitamin D-3 and its synthetic analogs inhibit the spontaneous in vitro immunoglobulin production by SLE-derived PBMC. Clinical Immunology 99:82-93. 35. Dusso, A.S., Brown, A.J., and Slatopolsky, E. 2005. Vitamin D. American Journal of Physiology-Renal Physiology 289:F8-F28. 36. Vernino, L., McAnally, L.M., Ramberg, J., and Lipsky, P.E. 1992. Generation of Nondividing High-Rate Ig-Secreting Plasma-Cells in Cultures of Human B-Cells Stimulated with Anti-Cd3-Activated T-Cells. Journal of Immunology 148:404-410. 37. Tangye, S.G., and Hodgkin, P.D. 2004. Divide and conquer: the importance of cell division in regulating B-cell responses. Immunology 112:509-520. 38. Shapiro-Shelef, M., and Calame, K. 2005. Regulation of plasma-cell development. Nature Reviews Immunology 5:230-242. 39. Calfon, M., Zeng, H.Q., Urano, F., Till, J.H., Hubbard, S.R., Harding, H.P., Clark, S.G., and Ron, D. 2002. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415:92-96. 40. Cohen-Lahav, M., Shany, S., Tobvin, D., Chaimovitz, C., and Douvdevani, A. 2006. Vitamin D decreases NF kappa B activity by increasing I kappa B alpha levels. Nephrology Dialysis Transplantation 21:889-897. 41. Giarratana, N., Penna, G., Amuchastegui, S., Mariani, R., Daniel, K.C., and Adorini, L. 2004. A vitamin D analog down-regulates proinflammatory chemokine production by pancreatic islets inhibiting T cell recruitment and type 1 diabetes development. Journal of Immunology 173:2280-2287. 42. Munker, R., Kobayashi, T., Elstner, E., Norman, A.W., Uskokovic, M., Zhang, W., Andreeff, M., and Koeffler, H.P. 1996. A new series of vitamin D analogs is highly active for clonal inhibition, differentiation, and induction of WAF1 in myeloid leukemia. Blood 88:2201-2209. 43. Wang, Q.M., Chen, F., Luo, X., Moore, D.C., Flanagan, M., and Studzinski, G.P. 47 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, ,,,参考文献 ,,,,,,,, 1998. Lowering of p27(Kip1) levels by its antisense or by development of resistance to 1,25-dihydroxyvitamin D-3 reverses the G1 block but not differentiation of HL60 cells. Leukemia 12:1256-1265. 44. Liu, W., Asa, S.L., and Ezzat, S. 2002. Vitamin D and its analog EB1089 induce p27 accumulation and diminish association of p27 with Skp2 independent of PTEN in pituitary corticotroph cells. Brain Pathology 12:412-419. 45. Dackiw, A.P.B., Ezzat, S., Huang, P., Liu, W., and Asa, S.L. 2004. Vitamin D3 administration induces nuclear p27 accumulation, restores differentiation, and reduces tumor burden in a mouse model of metastatic follicular thyroid cancer. Endocrinology 145:5840-5846. 46. Li, P.F., Li, C.R., Zhao, X.H., Zhang, X.H., Nicosia, S.V., and Bai, W.L. 2004. P27(Kip1) stabilization and G(1) arrest by 1,25-dihydroxyvitamin D-3 in ovarian cancer cells mediated through down-regulation of cyclin E/cyclin-dependent kinase 2 and Skp1-Cullin-F-box Protein/Skp2 ubiquitin ligase. Journal of Biological Chemistry 279:25260-25267. 47. Morse, L., Chen, D.Q., Franklin, D., Xiong, Y., and ChenKiang, S. 1997. Induction of cell cycle arrest and B cell terminal differentiation by CDK inhibitor p18(INK4c) and IL-6. Immunity 6:47-56. 48. Schrantz, N., Beney, G.E., Auffredou, M.T., Bourgeade, M.F., Leca, G., and Vazquez, A. 2000. The expression of p18(INK4) and p27(kip1) cyclin-dependent kinase inhibitors is regulated differently during human B cell differentiation. Journal of Immunology 165:4346-4352. 49. Nesby-O'Dell, S., Scanlon, K.S., Cogswell, M.E., Gillespie, C., Hollis, B.W., Looker, A.C., Allen, C., Doughertly, C., Gunter, E.W., and Bowman, B.A. 2002. Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age: third National Health and Nutrition Examination Survey, 1988-1994. American Journal of Clinical Nutrition 76:187-192. 50. McCarty, D.J., Manzi, S., Medsger, T.A., Ramseygoldman, R., Laporte, R.E., and Kwoh, C.K. 1995. Incidence of Systemic Lupus-Erythematosus - Race and Gender Differences. Arthritis and Rheumatism 38:1260-1270. 51. James, J.A., McClain, M.T., Arbuckle, M.R., and Harley, J.B. 2004. Autoantibodies before the clinical onset of systemic lupus erythematosus - The authors reply. New England Journal of Medicine 350:305-305. 52. Manz, R.A., Arce, S., Cassese, G., Hauser, A.E., Hiepe, F., and Radbruch, A. 2002. Humoral immunity and long-lived plasma cells. Current Opinion in Immunology 14:517-521. 53. Anolik, J.H., Campbell, D., Felgar, R., Rosenblatt, J., Young, F., and Looney, R.J. 2002. B lymphocyte depletion in the treatment of systemic lupus (SLE): Phase I/II trial of Rituximab (Rituxan (R)) in SLE. Arthritis and Rheumatism 46:S289-S289. 54. Renaudineau, Y., Pers, J.O., Bendaoud, B., Jamin, C., and Youinou, P. 2004. Dysfunctional B cells in systemic lupus erythematosus. Autoimmunity Reviews 3:516-523. 55. Dorner, T., and Lipsky, P. 2004. Correlation of circulating CD27(high) plasma cells and disease activity in systemic lupus erythematosus. Lupus 13:283-289. 56. Vaisberg, M.W., Kaneno, R., Franco, M.F., and Mendes, N.F. 2000. Influence of 48 上海交通大学,, 届博士论文,,,,,,,,,,,,,,,,,,, ,,,参考文献 ,,,,,,,, cholecalciferol (vitamin D3) on the course of experimental systemic lupus erythematosus in F1 (NZBxW) mice. Journal of Clinical Laboratory Analysis 14:91-96. 49 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 附件一 Modulatory Effects of 1,25-Dihydroxyvitamin D3 on Human B Cell Differentiation 1,2 1,3 Sheng Chen , Gary P. Sims , Xiao Xiang Chen 2 2 1 2 Yue Ying Gu , Shunle Chen and Peter E. Lipsky 1Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892 2Shanghai Clinical Center for Rheumatology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200001,China 3 Current address: Medimmune Inc. One Medimmune Way, Gaithersburg, MD 20878 Key words: 1,25-dihydroxyvitamin D3, B cell, Systemic lupus erythematosus Running Title: 1,25-dihydroxyvitamin D3 modulates B cells Scientific heading: Immunobiology Word count: Abstract: 200 words , Total Text: 9818 words Abstract: 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus had decreased 1,25(OH)2D3 levels, especially those with anti-nuclear antibodies and increased disease activity, suggesting that Vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)2D3 on B cell responses, and found that it inhibited ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and post-switch memory B cells was significantly inhibited by 1,25(OH)2D3, although upregulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in Vitamin D activity, 50 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 including 1α-hydroxylase, 24-hydroxylase and the Vitamin D receptor, each of which was regulated by 1,25(OH)2D3 and/or activation. Importantly, 1,25(OH)2D3 upregulated expression of p27, but not p18 and p21, that may be important in regulating proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)2D3 may play an important role in the maintenance of B cell homeostasis, and that correction of Vitamin D deficiency may be useful in the treatment of B cell mediated autoimmune disorders. Introduction 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the biologically active form of Vitamin D3 that was originally described as an essential hormone for bone and mineral homeostasis, but also has been shown to have immunomodulatory effects (1). Vitamin D3 can be derived from dietary sources, but the main source of Vitamin D3 is obtained through photosynthesis in the skin. Exposure of skin to ultraviolet (UV) light (270–300 nm) catalyzes the first step in the Vitamin D3 biosynthesis, converting 7-dehydroxycholesterol into pre-Vitamin D3, which is followed by a spontaneous and temperature dependent isomerization into Vitamin D3. To obtain the biologically active metabolite 1,25(OH)2D3, Vitamin D3 must first be hydroxylated by D3-25-hydroxylase (CYP2D25) in the liver into 25-hydroxyvitamin D3 (25(OH)D3). Further hydroxylation of 25(OH)D3 by 25(OH)D3-1-α-hydroxylase (CYP27B1) occurs mainly in the proximal convoluted tubule cells of the kidney, resulting in the biologically active 1,25(OH)2D3 (2). In addition, CYP27B1 is expressed by activated macrophages and dendritic cells (3, 4), but their contribution to systemic Vitamin D metabolism is unknown. 1,25(OH)2D3 also induces the expression of the major 1,25(OH)2D3 degrading enzyme, 24-hydroxylase (CYP24A1)(2). Most of the known biological effects of 1,25(OH)2D3 are mediated through the Vitamin D3 receptor (VDR), a member of the superfamily of nuclear hormone receptors (5) that is expressed by different cells of the immune system and elsewhere (5,6). The immunoregulatory effects of 1,25(OH)2D3 are mainly thought to be mediated through its action on antigen-presenting cells, with the most potent reported effects on dendritic cells (DCs) (7, 8). The in vitro differentiation of DCs from monocytes or murine bone 51 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 marrow-derived precursors is inhibited by 1,25(OH)2D3. Moreover, the antigen-presenting function of monocytes and DCs is profoundly inhibited, as is surface expression of costimulatory molecules, as well as IL-12 production (9). In addition, expression of the immunosuppressive cytokine IL-10 by DCs, that opposes the Th1 inducing effects of IL-12, is increased by 1,25(OH)2D3 (7, 8). Direct effects of 1,25(OH)2D3 on T lymphocytes have also been reported. T cell proliferation and cell cycle progression from G1a to G1b are inhibited in vitro by 1,25(OH)2D3. Cytokine production by T cells is also modulated, with inhibition of the Th1 cytokine IFN-γ, and an increase of Th2 cytokines, IL-4, IL-5 and IL-10 (10). Thus, 1,25(OH)2D3 is thought to polarize activated T cells towards a Th2 phenotype. In addition, it has also been shown that the expression of FasL by activated T cells could be repressed by 1,25(OH)2D3 (11). The effects of l,25(OH)2D3 on B cell function has not been examined in detail. Published reports have yielded conflicting observations. It has been claimed that 1,25(OH)2D3 may indirectly inhibit B cell function as a consequence of the impairment of CD4 T cell responses or the inhibition of cytokine production by monocytes/macrophages (12, 13). No direct effect of 1,25(OH)2D3 on B cell function has been reported. Interestingly, decreased 1,25(OH)2D3 serum concentrations have been reported in many autoimmune diseases, including systemic lupus erythmatosus (SLE) (14, 15). SLE is an autoimmune disease characterized by immune dysregulation resulting in over production of autoantibodies (16). Although the exact cause of SLE remains unknown, recent studies including the demonstration of the effectiveness of therapeutic B cell depletion, strongly implicate B cells as central players in the pathogenesis of this disease (17-20). We therefore, hypothesized that 1,25(OH)2D3 may be important in maintaining B cell homeostasis and that deficiency of 1,25(OH)2D3 might contribute to B cell hyperactivity in SLE. In this study, we measured 1,25(OH)2D3 and 25(OH)D3 levels in patients with SLE and correlated them with disease activity and the presence of anti-nuclear antibodies (ANA). 52 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 Subsequently, we investigated the direct effects of 1,25(OH)2D3 on primary human B cells to gain more insight into the potential role of Vitamin D on autoimmune disease pathogenesis. Our results provide evidence that Vitamin D might be a useful alternative therapy for the B cell hyperactivity characteristic of SLE. Results SLE patients have significantly diminished levels of 25(OH) Dand 1,25(OH)2D3 A total of 112 SLE patients who fulfilled ACR revised criteria (21) were assessed. Their characteristics are shown in supplemental Table 1. The serum 25(OH)D level was significantly lower in SLE patients (11.5 ? 1.5 ng/ml, N=57) than demographically comparable healthy controls (59.2 ? 6.5ng/ml, N=28) and RA patients (54.6 ? 5.2 ng/ml, N=27) (p<0.001) (Figure 1A). Notably, the level of 25(OH)D in newly diagnosed SLE patients without any treatment was 11.6 ? 2.1 ng/ml (N=12), which was not statistically different than that in SLE patients with established disease (11.8 ?1.8 ng/ml, N=44)(p>0.05) (Figure 1B). No correlation was found between the level of 25(OH)D and various clinical parameters including renal involvement, disease activity assessed by SLEDAI (22) and glucocorticoid use. The serum level of 1,25(OH)2D3 in SLE patients (14.5 ?1.2 pg/ml, N=87) was also significantly lower than in normal controls (29.8 ?1.5 pg/ml, N=30, p<0.001) (Figure 1C). 1,25(OH)2D3 levels were significantly lower in patients with a SLEDAI score >4 (12.2 ?1.6 pg/ml, N=52) compared to those with a SLEDAI score ? 4 (19.4 ? 1.9 pg/ml, N=36, P<0.001) (Figure 1D). Additional analysis demonstrated that the level of 1,25(OH)2D3 was significantly lower in patients with anti-nuclear antibody (ANA) compared with those without ANA (14.4 ? 1.3 pg/ml, N=71 versus 22.0 ? 4.2 pg/ml, N=11, p<0.05), but no significant difference was observed in patients with or without anti-dsDNA (15.0 ?1.8 pg/ml, N=39 versus 13.5 ? 1.7 pg/ml, N=43, p>0.05) (Figure 1E,F). The data indicated that patients with SLE had decreased levels of 25(OH)2D3 and 1,25(OH)2D3, and the latter was related to disease activity and ANA production. These results prompted us to examine the impact of Vitamin D on B cell function. 53 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 1,25(OH)2D3 inhibits proliferation of activated B cells Initially, the effect of 1,25(OH)2D3 on B cell survival was examined. 1,25(OH)2D3 did not affect the survival of unstimulated B cells in culture (Figure 2A). We next assessed the effect of 1,25(OH)2D3 on B cell proliferation. As shown in Figure 2B, 1,25(OH)2D3 exerted a significant inhibitory effect on B cell proliferation, especially in cultures activated with a combination of stimuli, e.g. anti-IgM/anti-CD40 (p<0.05), anti-CD40/IL-21 or anti-IgM/anti-CD40/IL-21 (p<0.01) . B cells stimulated with either anti-CD40 or anti-IgM alone had a limited proliferative capacity, and as a consequence the effect of 1,25(OH)2D3 was less consistent. CFSE labeling was also used to examine the effect of 1,25(OH)2D3 on proliferation of B cells. After 3 days in culture, some CFSE dilution was observed. Notably, cultures with and without 1,25(OH)2D3 exhibited a similar frequency of cells with modestly diluted CFSE (Figure 2C). As the culture period was prolonged, cells with increasing dilution of CFSE were observed. However, the extent of CFSE dilution was strikingly diminished in 1,25(OH)2D3 containing cultures compared to control cultures. These results were consistent with the conclusion that 1,25(OH)2D3 had little effect on initial cell division, whereas ongoing proliferation of stimulated B cells was suppressed. 1,25(OH)2D3 induces apoptosis of activated B cells To determine whether the inhibitory effect of 1,25(OH)2D3 on proliferation was associated with induction of apoptosis, we examined the percentage of apoptotic cells by Annexin V/PI staining. During 4 days in culture without stimulation, 1,25(OH)2D3 treatment had no significant effect on survival or apoptosis (Figure 3A). Stimulation with anti-IgM/anti-CD40 decreased the frequency of apoptotic cells, but in cultures of activated B cells, 1,25(OH)2D3 significantly increased the percentage of early apoptotic cells at day 4 (27.5% vs. 37.5%, p<0.01). This was time dependent as this difference was not apparent at earlier time-points (Figure 3B). 54 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 VDR and 24-hydroxylase can be induced by 1,25(OH)2D3 on B cells We next examined the expression of the Vitamin D receptor (VDR) and the known Vitamin D responsive gene, 24-hydroxylase (CYP24A1) by quantitative PCR. Freshly isolated B cells constitutively expressed very low levels of VDR mRNA. Compared to day 0 expression, the mean fold increase of VDR expression by B cells after 3 days culture with anti-IgM/anti-CD40, anti-CD40/IL21, anti-IgM/anti-CD40/IL21 or without stimulation was 1.45, 2.88, 4.44 and 1.46 fold respectively. 1,25(OH)2D3 increased VDR expression in both activated and unstimulated B cells (Figure 4A), but not earlier than 3 days (data not shown). In contrast, CYP24A1 expression was not detectable in either resting or activated B cells. However, it was significantly induced by 1,25(OH)2D3 (Figure 4B). The results demonstrate that VDR is expressed and inducible in primary B cells. Activation signals or 1,25(OH)2D3 upregulated VDR expression whereas CYP24A1 was only induced by 1,25(OH)2D3 but not by stimulation. 1,25(OH)2D3 specifically increases CD38 expression on B cells It was previously reported that 1,25(OH)2D3 could increase CD38 expression by lymphocytes (23, 24). To confirm this and to examine the effect of 1,25(OH)2D3 on other B cell markers, we activated purified B cells with various stimuli with and without 1,25(OH)2D3, and then stained the cells for various surface markers. The expression of CD38 on activated B cells was significantly increased by 1,25(OH)2D3. This difference was not observed with unstimulated B cells or cultures activated with anti-CD40 or anti-IgM alone, but it was apparent in B cells stimulated with the combination of anti-IgM/anti-CD40, anti-CD40/IL-21 or anti-IgM/anti-CD40/IL-21. In contrast, expression of IgD, CD21, CD23, CD27, CD86 and HLA-DR was altered by activating stimuli, but was not further affected by 1,25(OH)2D3 (Figure 5A). Similarly, 1,25(OH)2D3 had no effect on expression of CD19 or CD20 (data not shown). In order to examine the expression of CD40, we used anti-IgM and IL-4 to activate B cells and upregulate CD40 expression. Using these conditions, 1,25(OH)2D3 again increased CD38 expression (data 55 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 not shown), but CD40 expression was not altered by 1,25(OH)2D3. These results show that 1,25(OH)2D3 specifically increases CD38 expression by activated B cells, but has no effect on expression of a variety of other lineage, differentiation or activation markers. Increased expression of CD38 is commonly used as a phenotypic marker of plasma cells (25). To determine whether the 1,25(OH)2D3 mediated increase in CD38 expression was associated with plasma cell differentiation, we employed a method of in vitro stimulation known to induce plasma cells from peripheral B cell (anti-CD40 and IL-21 with or without anti-IgM) and examined the effect of 1,25(OH)2D3 (26). The induction of CD38 resulting from 1,25(OH)2D3 was not associated with an increase in CD27 expression, and, indeed, fewer CD38hi, CD27hi plasma cells were generated in the presence of 1,25(OH)2D3 (Figure 5B). Both the increase of CD38 expression and decrease of plasma cell generation occurred in a concentration-dependent manner, with the maximum effect noted reproducibly at 10 nM or less (Figure 5C). 1,25(OH)2D3 inhibits plasma cell differentiation and Ig secretion A more detailed analysis showed that 1,25(OH)2D3 inhibited plasma cell differentiation and the secretion of Ig (Figure 6). The generation of plasma cells was inhibited throughout the culture period after day 5 and 1,25(OH)2D3 caused significant decreases in both the percentage (Figure 6A, left panel) and absolute number of plasma cells (data not shown). In multiple experiments, the generation of phenotypically defined CD19+/- CD38hi CD27hi plasma cells was inhibited by 23-34% (Figure 6A, right panel). Analysis of culture supernatants revealed that 1,25(OH)2D3 also significantly decreased the secretion of Ig. Stimulation with anti-CD40 and IL-21 with or without anti-IgM, induced considerable Ig secretion by day 5 that increased through day 9 (Figure 6B). 1,25(OH)2D3 significantly inhibited IgG secretion. The percent inhibition progressively increased as the cultures were prolonged with the maximal inhibition noted at day 9 (p<0.001). A significant, albeit smaller, reduction in IgM production in 1,25(OH)2D3 cultures was only apparent at day 9. The effect of 1,25(OH)2D3 on the number of Ig secreting cells generated in these cultures was determined by ELISPOT assay. In agreement with the phenotype and 56 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 Ig production data, 1,25(OH)2D3 significantly reduced the number of IgG secreting cells in cultures of activated B cells. The number of IgM secreting cells was also significantly decreased by 1,25(OH)2D3 in anti-CD40/IL-21 cultures (Figure 6C). Notably, when 1,25(OH)2D3 was added at day 5 of culture, no effect on maintenance of plasma cells or Ig secretion was noted over the next 5 days (Figure 6D). This result indicates that 1,25(OH)2D3 inhibits the generation of plasma cells but not their subsequent persistence or Ig secretion. 1,25(OH)2D3 inhibits generation of post-switched memory B cells from naïve B cells To determine whether the inhibitory effect of 1,25(OH)2D3 was specific for plasma cell generation, we also examined its role in regulating the generation of post-switch memory B cells. To address this, naïve B cells (CD19+, IgG-CD27-) were purified by sorting (>99% purity) as shown in Figure 7A, and stimulated in a manner known to induce class-switch recombination(26). As shown in Figure 7B, the combination of anti-CD40 and IL-21 with or without anti-IgM was able to induce post-switched cells expressing surface IgG. In the absence of IL-21, few cells expressed surface IgG. Notably, the frequency of IgG positive cells was significantly lower in the 1,25(OH)2D3 containing cultures (Figure 7B,C), whereas there were more B cells expressing surface IgM (Figure 7B). This reduction in class-switching correlated with reduced plasma cell numbers and IgG production in parallel 7 day cultures (Figure 7D). These data demonstrate that 1,25(OH)2D3 inhibits generation of both post-switch memory cells and plasma cells. 1,25(OH)2D3 downregulates the expression of XBP1 and ERN1 mRNA, but not PAX5, BCL6, AID, BLIMP1, MTA3 and IRF4 Thus far, the results clearly demonstrated that 1,25(OH)2D3 reduced B cell proliferation, generation of post-switched memory B cells, plasma cell differentiation, and Ig secretion. However, the stage at which 1,25(OH)2D3 exerts its effects remained unclear. To gain further insight into the nature of B cell inhibition by 1,25(OH)2D3, we examined the expression of key transcription factors involved in B cell maturation and plasma cell differentiation. We compared the expression of genes involved in germinal center reactions, 57 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 including PAX5, BCL6, and AID as well as those involved in plasma cell differentiation, including BLIMP1, IRF4, MTA3, XBP1 and ERN1 (27). All the analyses were carried out at day 3 before significant effects on the frequency of memory B cells and plasma cells or Ig secretion were evident. Interestingly, 1,25(OH)2D3 had no significant effect on expression of PAX5, BCL6, AID, BLIMP1, MAT3 or IRF4. However, even at this relatively early time point expression of XBP1 and ERN1 was significantly reduced but not eliminated by 1,25(OH)2D3 (Figure 8). The lack of a consistent effect of 1,25(OH)2D3 on the genetic programs regulating the differentiation of memory cells or plasma cells suggests that it may inhibit their maturation by other means. 1,25(OH)2D3 increases the expression of p27 mRNA, but not P21 and P18 To determine the mechanism involved in the inhibitory effect of 1,25(OH)2D3 on B cells, we initially screened an array of NF-κB mediated signal transduction pathway genes. As shown in Supplement Figure 2, a few mRNAs were up- or down-regulated by 1,25(OH)2D3, but no general pattern of gene regulation was observed consistent with a major effect on the NF-κB signaling pathway. We next examined the effect of 1,25(OH)2D3 on cell cycle related gene expression by B cells (shown in Supplement Figure 2). We found a number of genes, including as cyclin D1, cyclin D2, cyclin T1, cyclin T2, cyclin-dependent kinase 4 and cyclin-dependent kinase 6 were significantly downregulated by 1,25(OH)2D3. In contrast, cyclin dependent kinase inhibitor p27 was significantly upregulated by 1,25(OH)2D3, whereas most genes, including cyclin dependent kinase inhibitor p21 remained unchanged. We verified these findings using quantitative PCR and found that p27, but not p21 or p18 mRNA was significantly upregulated by 1,25(OH)2D3 in activated B cells (Figure 9). B cells can directly metabolize 25(OH)D3 into a biologically active molecule It has been shown that the precursor of 1,25(OH)2D3 , 25(OH)D3, is present in serum and is converted to the active form by 1α-hydroxylase (CYP27B1), which is mainly expressed in kidney and also can be found in monocytes/macrophages (3, 4). The expression of 1α-hydroxylase mRNA by human peripheral B cells was therefore investigated by RT-PCR. 58 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 As shown in Figure 10A, B cells constitutively expressed 1α-hydroxylase mRNA and it was upregulated following stimulation, but not further induced by 1,25(OH)2D3. To determine whether this upregulation had a functional consequence, we added the precursor, 25(OH)D3, to purified B cells stimulated with anti-CD40 and IL-21 and examined the expression of CD38, plasma cell differentiation and IgG production. 25(OH)D3 increased CD38 expression, and inhibited plasma cell generation and IgG production in a concentration dependent manner (Figure 10B). The precursor had similar effects to the active form of Vitamin D3, but at a 25-fold higher concentration. Since monocytes contaminating the peripheral B cell populations might contribute to these results, B cells that were sorted from tonsils and lacked detectable monocytes/macrophage contamination were assessed and similar results obtained (data not shown). There results are consistent with the conclusion that human B cells can take up and metabolize 25(OH)D3 to active 1,25(OH)2D3 that can inhibit B cell function. Discussion Here we show that 1,25(OH)2D3 has potent direct effects on B cell responses inhibiting proliferation, generation of class-switched memory B cells, plasma cell differentiation and Ig production. Reduced levels of 1,25(OH)2D3 in SLE patients, particularly in those patients with high disease activity scores and ANAs, suggest that Vitamin D dependent B cell regulation may play an important role in maintaining normal B cell homeostasis and that decreased levels of 1,25(OH)2D3 may contribute to B cell hyperactivity in SLE. Finally, since activated B cells express many of the molecules involved in Vitamin D metabolism, their characteristic hyperactivity in SLE could contribute to the decreased levels of 1,25(OH)2D3 found in these patients. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active metabolite of Vitamin D3, not only regulates bone and calcium metabolism but also exerts important effects on the immune system. These effects have been attributed to an action on antigen presenting cell (mainly dendritic cells) and activated T cells (7-10). Previously, it was widely believed that 59 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 the inhibitory effect on B cell function was indirect and mediated through the inhibition of CD4 T cell responses or inhibition of cytokine production by monocytes/macrophages (12, 28). However, the current studies clearly show a direct inhibitory effect of Vitamin D on B cell function. Importantly, the levels of 1,25(OH)2D3 found to downregulate B cell function in this study were well within serum levels in normal humans (29, 30). The varied functions of 1,25(OH)2D3 are mainly mediated through binding to the VDR. We clearly demonstrate that VDR mRNA is constitutively expressed in human primary B cells at low levels and is upregulated following stimulation and enhanced in the presence of 1,25(OH)2D3 in a time-dependent manner. Previously the expression of VDR on B cells has been controversial. It has been claimed that B cells do not contain detectable amounts of VDR, and the highest levels of VDR are expressed by CD8 and CD4 lymphocytes (13), However, it has also been reported that VDR is constitutively expressed on human tonsil B cells and can be further upregulated by activation (24), and that VDR is expressed on B cell lymphoma cell lines SUDHL4 and SUDHL5 (31). Our data analyzing the expression of VDR mRNA and also the functional activity of 1,25(OH)2D3 clearly show that the VDR is expressed by human peripheral B cells. Moreover, our data clearly demonstrate that VDR expression by B cells is regulated both by 1,25(OH)2D3, as it is on other cells (5), but also by activation signals. These results indicate that Vitamin D may exert differential effects on activated versus resting B cells and also may have different effects in individuals with different levels of serum 1,25(OH)2D3. Active 1,25(OH)2D3 is mainly degraded by 24-hydroxylase (CYP24A1) (2). Microarray analysis from human colon carcinoma and ovarian cancer cell lines revealed that CYP24A1 was the most inducible gene responsive to 1,25(OH)2D3 (32, 33). Here we show that 24-hydroxylase was significantly upregulated following the incubation of human B cells with 1,25(OH)2D3. As opposed to the VDR, CYP24A1 was not altered by B cell activation. These results further demonstrate that human B cells can respond to 1,25(OH)2D3 directly. In addition, the results suggest that the activity of Vitamin D on B cells might be influenced not only by VDR expression but also by the capacity to degrade the active molecule. The 60 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 increased susceptibility of activated B cells to many of the effects of Vitamin D might therefore reflect the upregulation of VDR but not CYP24A1 by these cells. The possible relevance of these findings to immunoregulation in SLE is supported by a previous study that showed 1,25(OH)2D3 inhibited B cell responses in mixed PBMC cultures from SLE patients (34). 25(OH)D3-1-α-hydroxylase (CYP27B1), the enzyme responsible for the final hydroxylation and activation of 25(OH)D3 into 1,25(OH)2D3, is mainly found in the proximal convoluted tubule cells of the kidney (2). Interestingly, we found CYP27B1 mRNA was also expressed by resting B cell and could be further induced by stimulation, but not by 1,25(OH)2D3. Moreover, we found that the precursor, 25(OH)D3, had similar effects on purified B cell compared to the active form, albeit at higher concentrations. Therefore, 25(OH)D3 might be metabolized to 1,25(OH)2D3 by B cells themselves and may represent a source for extra-renal synthesis of 1,25(OH)2D3 , as is thought to be the case for macrophages and dendritic cells (3, 4). In conditions such as SLE, in which there is diffuse B cell activation (20), systemic Vitamin D metabolism might therefore be significantly influenced. The capacity of 1,25(OH)2D3 to inhibit proliferation has been reported in lymphocytes and a variety of human cancer cell lines (35). We showed that 1,25(OH)2D3 also exerted an inhibitory effect on B cell proliferation. By using CFSE analysis, initial B cell division was not affected by 1,25(OH)2D3 whereas subsequent ongoing proliferation was progressively inhibited. This effect coincides with activation and 1,25(OH)2D3 mediated upregulation of the VDR and suggests that a threshold level of VDR engagement might be required for the anti-proliferative effect of the occupied VDR to become apparent. Notably, 1,25(OH)2D3 mediated inhibition of proliferation was associated with apoptosis of the activated and dividing B cells. These results suggest that differentiative events that require initial expansion of B cells might be eliminated as a result of the 1,25(OH)2D3 mediated death of proliferating B cells. 61 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 The inhibition of proliferation and induction of apoptosis are likely to result in the significant reduction in plasma cell differentiation and immunoglobulin production that we observed. Previous studies have shown that plasma cell differentiation requires initial B cell proliferation (36) and increases with the number of cell divisions (37). We have previously shown that the combination of IL-21 and anti-CD40 stimulation with or without BCR-crosslinking is a potent inducer of proliferation and plasma cell differentiation and that all the plasma cells generated had undergone extensive proliferation (26). Using this system, we demonstrated that 1,25(OH)2D3 had inhibitory effects on plasma differentiation and Ig production. Interestingly, this effect is not evident when the B cells are treated with 1,25(OH)2D3 after 5 days in culture, indicating that 1,25(OH)2D3 inhibits the generation of plasma cells but not their subsequent persistence and lends support to the conclusion that the inhibition of B cell proliferation by 1,25(OH)2D3 is responsible for the reduction of Ig-secreting cells and Ig production. Several transcription factors regulate different stages of B cell maturation and plasma cell differentiation(38). The most notable finding of the current study was that expression of most of these transcriptional regulators was unaffected by 1,25(OH)2D3, including PAX5, BCL6, AID, BLIMP1, MTA3 and IRF4. These results provide no support for the possibility that 1,25(OH)2D3 inhibits memory cell or plasma cell differentiation by directly affecting expression of any of these regulator of B cell maturation. However, the expression of XBP1 was modestly, but significantly, downregulated by 1,25(OH)2D3. In addition, mRNA for ERN1, which is required for processing XBP1 mRNA to a spliced form that encodes a more stable and active protein (39), was also downregulated by 1,25(OH)2D3. The inhibitory effect of 1,25(OH)2D3 on expression of XBP1 and ERN1 mRNA levels may explain the greater inhibitory effect of Vitamin D on Ig secretion and detection of plasma cells by ELISPOT compared to its more modest inhibition of the differentiation of phenotypically defined plasma cells. Although inhibition of expression of XBP1 and ERN1 mRNA by 1,25(OH)2D3 may contribute to a reduced amount of Ig secretion per phenotypically defined plasma cell, it is unlikely to explain the inhibition of the generation of both plasma cells and post-switched memory cells as well as ongoing 62 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 proliferation of activated B cells by Vitamin D. Recent studies have shed some light into the molecular basis of the immunomodulatory activity of 1,25(OH)2D3. Several important intracellular pathways have been reported to be inhibited by 1,25(OH)2D3, such as the NF-κB activation and cell cycle progession. The suppressive effect of 1,25(OH)2D3 on the NF-κB signaling pathway was previously observed in T cells, monocytes or macrophages (9, 40,41), and could have affected expression of various essential cell surface and secreted molecules. The NF-κB pathway, however, did not appear to be generally suppressed by 1,25(OH)2D3 in activated B cells as assessed by analysis of expression of various genes influenced by this signaling pathway. Since 1,25(OH)2D3 directly inhibited proliferation of activated B cells, we therefore examined whether Vitamin D might suppress expression of a variety of cell cycle regulators, and found that 1,25(OH)2D3 increased expression of p27 and decreased mRNA levels of CDK4 and 6 and cyclin D. Cell cycle progression is under the control of cyclin dependent kinases (CDK), the activity of which is dependent on the expression of specific CDK inhibitors. Previous studies have shown that 1,25(OH)2D3 inhibits proliferation of cancer cells by inducing gene transcription of CDK inhibitors p21 and/or p27, which can inhibit cell cycle progression at the G1/S transition, depending on the cell type (42 -44). In vivo, 1,25(OH)2D3 administration can effectively restore p27 accumulation in cancer cells as detected by immunohistochemistry, and reduce the tumor burden (45). Several mechanisms have been reported to be involved, including increasing p27 transcription mediated by VDR-Sp1 interactions with the p27 promoter (46), stabilization of the protein through VDR-induced reduction of CDK2 activity and Skp2 abundance (the main factors responsible for p27 degradation), or induction of PTEN, a phosphatase that dephosphorylates p27 (44). Other CDK inhibitors have been shown to play an essential role in B cell responses. In this regard, p18 has been shown to be required for B cells to terminate proliferation and differentiate into functional plasma cells (42, 47, 48). Previous studies have confirmed that p18 and p27 were involved in regulating different steps of B proliferation. Whereas p27 was thought to regulate cell cycle entry by forming a ternary complex with CDKs and cyclins, p18 was directly involved in induction of the G1 cell cycle 63 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 arrest by blocking association of CDK4 and 6 with cyclin D (48). Interestingly, our data demonstrated that the mRNA level of p27, but not p21 or p18 was upregulated by 1,25(OH)2D3 in activated human B cells. These results suggest that 1,25(OH)2D3 could inhibit B cell proliferation by upregulating p27 and thereby inhibiting cell cycle entry of previously cycling B cells. Since 1,25(OH)2D3 also decreased mRNA levels of CDK4 and 6 as well as cyclin D, the effect of p27 on ongoing B cell proliferation might be more profound. The 1,25(OH)2D3 mediated induction of p27 may limit ongoing B cell proliferation and thereby play an important role in the control of B cell responses. These findings suggest that the major effects of Vitamin D on plasma cell and memory cell differentiation may be largely indirect and result from suppression of ongoing B cell proliferation which is required before these differentiation steps can occur(36). Decreased Vitamin D concentrations have been reported in many autoimmune diseases, such as multiple sclerosis, type 1 diabetes mellitus, RA, fibromyalgia and SLE (14, 15, 30). SLE is an autoimmune disease characterized by immune dysregulation resulting in the production of antinuclear antibodies (ANA), generation of circulating immune complexes, and activation of the complement system (16). Although the exact cause remains unknown, more recently studies including the demonstration of the effectiveness of therapeutic B cell depletion, strongly indicate that B cells play a central role in the pathogenesis of this disease (17-20). We examined serum levels of 25(OH)D3 and 1,25(OH)2D3 in Chinese lupus patients. Consistent with previous findings, both 25(OH)D3 and 1,25(OH)2D3 levels were significantly lower in patients with SLE compared to healthy donors (14), However, we also demonstrated that the lower levels of 1,25(OH)2D3 significantly correlated with increased disease activity. Moreover, significantly decreased Vitamin D levels were detected in 12 newly diagnosed patients before any treatment was administered, indicating that deficiency may be present at the onset of lupus and possibly before. This could explain, in part, why African Americans who are often chronically Vitamin D deficient (29, 49) also have a higher risk and severity of SLE (50). It is known that SLE may be preceded by autoantibody production that may exist for many years before diagnosis (51) and it is possible that Vitamin D deficiency may contribute to this. The current results are consistent 64 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 with the conclusion that Vitamin D is involved in maintaining normal B cell homeostasis and that Vitamin D deficiency in SLE patients may contribute to B cell hyperactivity, the breakdown of B cell tolerance and the generation of pathogenic autoantibodies. The association with decreased 1,25(OH)2D3 levels in lupus patients with positive ANA is consistent with this possibility. Notably, we also found that those SLE patients with positive ANA titers had lower levels of 1,25(OH)2D3 than those without ANA. Interestingly, such a difference was not seen between the patients who were positive or negative for dsDNA antibodies. The persistent and therapy-resistant presence of ANA in SLE even during the clinically quiescent phases has been explained by the existence of long-lived plasma cells, which are responsible for antibody-mediated autoimmune memory in the absence of any detectable memory B cells (52). In contrast, the presence of dsDNA antibody is usually correlated with SLE activity and decreases after immunosuppressive or B cell depletion therapy, suggesting that these autoantibodies originate from short-lived dividing plasmablasts (20, 53). Thus, 1,25(OH)2D3 may play a role in regulating the generation of long-lived plasma cell and be less potent in suppressing the rapid production of autoantibody from dividing plasmablasts. The kinetics of the in vitro inhibition of B cell responses by 1,25(OH)2D3 is consistent with this possibility. Besides the inhibitory effect on plasma cell differentiation and Ig production, our data also demonstrated that 1,25(OH)2D3 exerted its inhibitory effects on memory B cell differentiation. Relative increases in memory cells and plasma cells are both characteristic B cell abnormalities in lupus (54). In contrast to naïve B cells and plasma cells, memory B cells are not targeted by conventional immunosuppressive therapies and may contribute to disease flares (55). Thus, Vitamin D therapy may offer a new opportunity to induce remission in SLE by preventing or decreasing the differentiation of memory cells. This possibility is supported by murine studies in which administration of 1,25(OH)2D3 or its analog has been shown to decrease proteinuria or increase survival in a spontaneous mouse lupus model MRL/lpr (56). 65 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 In conclusion, 1,25-dihydroxyvitamin D3 may play an important role in the maintenance of B cell homeostasis. Correction of Vitamin D deficiency may be useful not only to prevent osteopenia but also in the suppression of B cell overactivity in autoimmune disorders, such as SLE. Methods Clinical samples For the analysis of serological levels of 25(OH)D and 1,25(OH)2D3, peripheral blood samples from patients with SLE, patients with rheumatoid arthritis (RA) and demographically comparable healthy controls were obtained from the Shanghai Clinical Center for Rheumatology, Renji Hospital, Shanghai, China after informed consent. The diagnosis of SLE relied on ACR revised criteria (21). Clinical charts of all patients were reviewed and disease activity was scored according to the SLEDAI (22). For the analysis of B cell function in vitro, blood samples from normal healthy adult donors were obtained from the Warren G. Magnuson Clinical Center Blood Bank. Collection of samples was approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institute of Diabetes & Digestive & Kidney Diseases (NIAMS/NIDDK) Institutional Review Board, and informed consent was obtained according to the Declaration of Helsinki. Determination of serum 25(OH)D and 1,25(OH)2D3 levels Serum total 25(OH)D and 1,25(OH)2D3 levels were measured in SLE patients, patients with RA and healthy donors by radioimmunoassay assays (RIA) or by Enzyme Linked ImmunoSorbent Assay ELISA (both from Alpco Diagnostics, Windham, NH) according to manufacturer's instructions. Both assays yielded similar results. B cell enrichment, flow cytometry and cell sorting. B cells were enriched by negative selection from buffy coats or leukapheresis samples using RosetteSep or StemSep B cell purification antibody cocktails (Stem Cell Technology, Vancouver, Canada). Purified B cells were stained with various mAb combinations for 20 66 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 min on ice in staining buffer (1%BSA, 5%FCS in PBS). The directly conjugated mAb used were anti-IgD-FITC, anti-CD27-PE, anti-CD40-PE, anti-CD86-FITC, anti-IgG-PE, anti-CD19-APC, anti-HLA-DR-PE, (Becton Dickinson Pharminogen, San Diego, CA), anti-CD19-PerCpCy5.5, anti-CD38-APC (clone HB7) (Becton Dickinson Immunocytometry, San Jose, CA). Stained cells were washed and data were collected immediately using a four-color FACScalibur (Becton Dickinson) or fixed in 1% paraformaldehye and analyzed within 24 h. Data were analyzed using FlowJo software (TreeStar, Stanford University, Palo Alto, CA). B cell populations were sorted using the Dako Cytomation MoFlo (Dakocytomation, Fort Collins, CO). Cell apoptosis and viability assay. To quantify apoptosis, cultured B cells were double-stained with Annexin V-FITC conjugate and propidium iodide (PI) using TACS Annexin V Kits from Oncogene (San Diego, CA, USA) according to the manufacturer’s directions. The stained B cells were immediately analyzed using a FACSCalibur flow cytometer (Becton Dickinson). B cell stimulation B cells were cultured in 96-well plates at 1x106 cells/ml in 100 µl volumes of culture medium (10% FCS in RPMI medium with L-glutamine, penicillin-streptomycin) alone or supplemented with various cytokines and stimuli; 1 µg/ml anti-CD40 (R&D Systems, Minneapolis, MN), 3 µg/ml goat F(ab’)2 anti-IgM (Jackson ImmunoReseach, West Grove, PA), 250 ng/ml IL-21 (Cell Sciences, Canton, MA), 50 ng/ml IL-4 (R&D Systems, Minneapolis, MN), 1,25(OH)2D3 (usually 10 nM) and 25(OH) D3 (both from Sigma, Saint Louis, MO),. Proliferation Assay Cells were cultured for 3, 4 or 5 days as described above and pulsed for an additional 16 hours with 3H-thymidine (1µCi, 37KBq) and then harvested. The 3H-thymidine incorporation was measured with a Top Count microplate scintillation counter (Packard Instruments, Downers Grove, IL). Alternatively, proliferation was assessed by 67 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 carboxy-fluorescein diacetate, succinimidyl ester (CFSE, Invitrogen, Carlsbad, CA) dilution. Briefly, 1x107 B cells were incubated with 1 µM of CFSE for 15 minutes at room temperature, then quenched with an equal volume of 100% FCS for 2 minutes and washed. CFSE labeled cells were cultured for 3 to 6 days as mentioned above and data were collected by FACS analysis. Immunoglobulin ELISA Culture supernatants were incubated on goat anti-human IgG and IgM (Bethyl Laboratories, Montgomery, TX) coated Maxisorp plates (Nalge Nunc International, Rochester, NY), and bound Ig was detected using AP-conjugated goat anti-human IgG or IgM (Bethyl Laboratories) with Sigma Fast pNPP alkaline phosphate substrate (Sigma, St. Louis, MO). ELISPOT assay The number of IgG and IgM secreting cells was determined with the ELISPOT assay. MultiScreen-HTS plates (PVDF membrane, Millipore, Bedford, MA) were coated overnight at 4?C with 50 µl/well goat anti-human IgG or IgM (Bethyl Laboratories, Montgomery, TX) respectively, at a concentration of 5 µg/ml diluted in 0.05M carbonate phosphate-buffered saline (Sigma). After washing, unbound surfaces of wells were blocked with culture medium for 1 h at 25?C. Stimulated cells from 7 day cultures were washed three times and resuspended with medium containing 10% Ultra Low IgG FCS (Invitrogen). Serial dilutions of cells were added to wells in triplicate and incubated at 37?C overnight. Medium containing 10% Ultra Low IgG FCS was included in all experiments as a negative control. Thereafter, plates were washed with TBS/0.05% Tween20 four times and incubated with either biotinylated goat–antihuman IgM or IgG (Invitrogen) diluted 1:2000 in TBS/0.05% Tween20 at 25?C for 3 h. Plates were then washed in TBS/0.05% Tween20 and incubated with 0.5 µg/ml alkaline phosphatase-conjugated streptavidine (Sigma) in TBS/0.05% Tween20 for 1 h at 25?C. All plates were developed with alkaline phosphatase substrate Kit III (Vector Laboratories, Inc. Burlingame, CA) after washing in distilled water. Immunoglobulin-secreting cells appeared as blue spots, which were counted using a Cellular 68 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 Technology Ltd Series 3B Analyzer (CTL Analyzer LCC, Cleveland, OH). RNA preparation and quantitative RT-PCR. Total RNA from B cells was purified by using the Rneasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's manual. Reverse transcription reactions were prepared using the SuperScript One-Step PCR System with platinum Taq polymerase and ROX reference dye (Invitrogen Life Technologies). Fifty nanograms of isolated RNA was added per reaction with 1.2 mM MgSO4. TaqMan Assays on Demand Gene expression primer/probe sets for β2M (beta-2-microglobulin, Hs99999907_m1), VDR (Hs01045840_m1), CYP27B1 (Hs00168017_m1), CYP24A1 (Hs00167999_m1), Pax5 (paired box gene 5, Hs00277134_m1), Bcl6 (B-cell CLL/lymphoma 6 (zinc finger protein 51, Hs00153368_m1), Blimp1( B-lymphocyte-induced maturation protein 1, Hs00153357_m1), IRF4 (interferon regulatory factor 4, Hs00180031_m1), XBP1 (X-box binding protein 1, Hs00231936_m1),ERN1 (endoplasmic reticulum to nucleus signaling 1, Hs00176385_m1), AID (activation-induced cytidine deaminase, Hs00221068_m1), p27 (cyclin-dependent kinase inhibitor 1B, Hs00153277_m1), p21 (cyclin-dependent kinase inhibitor 1A, Hs00355782_m1) and p18 (cyclin-dependent kinase inhibitor 2C, Hs00176227_m1) were purchased from Applied Biosystems (Foster City, CA), and qRT-PCR reactions were performed according to the manufacturer’s instructions. mRNA expression for each gene was calculated in triplicate using the comparative cycle threshold method with efficiency calculations and all mRNA levels were normalized to β2M. The effects of 1,25(OH)2D3 on the expression of 84 cell cycle regulated genes and 84 genes associated with NF-κB mediated signal transduction were examined using Human Cell Cycle RT?Profiler? PCR Array and Human NFκB Signaling Pathway RT?Profiler? PCR Array respectively (SuperArray Bioscience, Frederick, MD) according to the manufacturer's instructions. In brief, cDNA was prepared from 1 µg total RNA by using a PT2 PCR array first strand kit. A total volume of 25 µl of PCR cocktail, which included 12.5 µl of RT Real-Time SYBR Green/ROX PCR Master Mix from SuperArray Bioscience 69 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 (Frederick, MD) (containing HotStart DNA polymerase, SYBR Green dye, and the ROX reference dye), 11.5 µl of double-distilled H2O and 1 µl of template cDNA was loaded in each well of the PCR array. PCR amplification was carried out with an initial 10 min step at 95?C, followed by 40 cycles of (95?C, 15 s; 60?C, 1 min). The fluorescent signal from SYBR Green was detected immediately after the extension step of each cycle and the cycle at which the product was first detectable was recorded as the cycle threshold. Data were imported into an Excel database and analyzed using the comparative cycle threshold method with normalization of the raw data to housekeeping genes including β2M, HPRT1 (Hypoxanthine phosphoribosyltransferase 1), RPL13A (Ribosomal protein L13a), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and ACTB (β-Actin). Analysis and Statistics Statistical significance of the data was analyzed with a two-tailed, nonpaired or paired, Student t test, using Microsoft Excel Windows software. Data are plotted as mean ? SEM. Asterisk (*) denotes p value <0.05, (**) denotes p value <0.01 and (***) denotes P value <0.001. Acknowledgments We gratefully acknowledge the assistance of the Flow Cytometry Section of the Office of Science and Technology, NIAMS for sorting B cell subsets. We are also grateful to Dr John A. Hardin for many stimulating discussions and to Dr Nancy Longo for reviewing the manuscript. This work was supported by funds from the NIAMS intramural research program. We declare there is no financial conflict of interest. Figure Legends Figure 1. Patients with SLE have decreased 25(OH)D and 1,25(OH)2D3 levels. Serum levels of 25(OH)D and 1,25(OH)2D3 levels were measured by RIA and ELISA. A. 25(OH)D levels in SLE patients (N=57) compared to normal controls (NC, N=28) and RA 70 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 patients (N=29). Mean levels in SLE patients were significantly different from those in NC and RA patients (P<0.001). B. 25(OH)D levels in patients with established SLE with a mean duration since diagnosis of 5.11 ? 0.88 years (N=45) compared to newly diagnosed, untreated patients with a mean duration of 25.8 ? 9.2 days (N=12). There was no significant difference in mean 25(OH)D levels in newly diagnosed or established SLE patients (p>0.05). C. 1,25(OH)2D3 levels in SLE patients (N=87) compared to normal controls (NC) (N=30). The 1,25(OH)2D3 levels were significantly lower in SLE patients (P<0.001). D. 1,25(OH)2D3 levels in non-active SLE patients (SLEDAI ?4, N=35) compared to active patients (SLEDAI >4,N=52). Active SLE patients had significantly lower levels of 1,25(OH)2D3 (P<0.001). E. 1,25(OH)2D3 levels in SLE patients with positive ANA (N=71) compared to those without ANA (N=11). Patients without ANA had significantly higher levels of1,25(OH)2D3 (P<0.05). F. 1,25(OH)2D3 levels in SLE patients with anti-dsDNA (N=39) compared to those without anti-dsDNA (N=43). 1,25(OH)2D3 levels did not differ between these groups. Data are shown as mean ? SEM and significant differences between the groups are shown by *** (p<0.001) or * (p<0.05). Figure 2. Vitamin D inhibits proliferation of human peripheral B cells. A. Purified B cells were cultured with or without 1,25(OH)2D3 (10 nM) and stained with Annexin V /PI. Percentages of viable cells (double negative for Annexin V and PI) are shown as mean ? SEM of triplicate cultures and are representative of three independent experiments. B. Purified B cells were stimulated with combinations of anti-IgM, anti-CD40 and IL-21 as indicated in the presence or absence of 1,25(OH)2D3 (10nM). After 3 days, cells were pulsed with 3H-thymidine and incorporation was measured 16 h later. Data are the mean ? SEM of triplicate cultures and are representative of three independent experiments. Significant differences between Vitamin D and control responses are shown by * (p<0.05) or ** (p<0.01). C. Purified B cells labeled with CFSE were cultured with anti-IgM, anti-CD40 and IL-21 in the presence or absence of 1,25(OH)2D3 (10 nM). At the time-points indicated, CD19+ B cells were analyzed for CFSE dilution. Data shown are representative of results from three experiments. 71 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 Figure 3. Vitamin D induces apoptosis of activated human peripheral B cells. Purified B cells were cultured with no stimulus, anti-IgM and anti-CD40 alone or combination in the presence or absence of 1,25(OH)2D3 (10 nM). A. After 4 days in culture, cells were washed and stained with Annexin V and PI and analyzed by flow cytometry. The numbers in the upper left quadrants represent the percentage of apoptotic cells in culture. B. Percentages of apoptotic cells in 3 and 4-day cultures with no stimulus or anti-IgM and anti-CD40 and with or without addition of 1,25(OH)2D3(10 nM). Data are mean ? SEM of triplicate determinations from one of three independent experiments. Asterisks indicate the statistical significance of the difference between Vitamin D and untreated B cells (p?0.01). Figure 4. Vitamin D inducible genes are upregulated on activated B cells. Purified B cells were stimulated in various conditions in the presence or absence of 1,25(OH)2D3 (10 nM) as indicated. Total RNA was extracted from cells at day 0 and after 3 days in culture. Gene expression was detected by quantitative RT-PCR in triplicate. A. Expression of VDR relative to β2M. Left panel shows results from stimulated versus fresh B cells, and the right panel shows results from B cells culture without stimulation. B. Expression of CYP24A1 relative to β2M. Left panel shows results from stimulated versus fresh B cells, and the right panel shows results from B cells cultured without stimulation. Data are the mean ? SEM from triplicates from one of two representative experiments. Significant differences between Vitamin D and control responses are shown by * (p<0.05) , ** (p<0.01) or *** (p<0.001). Figure 5. Vitamin D increases CD38 expression on activated B cells. A. Purified B cells were cultured with various stimuli in the presence or absence of 1,25(OH)2D3 (10 nM). After 4 or 5 days in culture, cells were washed and cell surface markers were analyzed by flow cytometry. The expression of IgD, CD27, CD38, CD21, CD23, HLA-DR, CD86 and CD40 was compared between Vitamin D (solid line) and control (dotted line) cultures. B. The plasma cells population based on the phenotype, CD19+/-, CD27hi, CD38hi (upper right quadrant) , was compared between cultures with or without 1,25(OH)2D3 (10 nM) after 7 days of culture with the stimuli indicated. C. Concentration dependent effect of 72 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 1,25(OH)2D3 (10 nM) on CD38 expression and plasma cell generation from B cells cultured with anti-CD40 and IL-21 for 7 days. Figure 6. The inhibitory effect of Vitamin D on plasma cell differentiation and Ig production. Purified B cells were cultured with anti-CD40 and IL-21 with or without anti-IgM in the presence or absence of 1,25(OH)2D3 (10 nM). A. The generation of plasma cells (CD19+/-, CD27hi, CD38hi) was assessed after 5, 7 and 9 days in culture (left panel). The frequencies of plasma cells in Vitamin D versus control cultures after a 7 day incubation of anti-CD40/IL-21 (n=12) and anti-IgM/anti-CD40/IL-21 stimulated B cells (n=8) were determined and shown as mean % of no Vitamin D control (right panel). B. IgG and IgM production determined by ELISA. The results are the mean?SEM and are representative of five independent experiments. Significant differences are shown as ** (p<0.01) or *** (p<0.001). C. IgG (left) IgM (right) secreting cells were determined at day 7 by ELISPOT. Data are the mean?SEM from triplicate determinations and are representative of one of three independent experiments. D. Purified B cells were cultured with anti-CD40 and IL21 for 5 days, after which 1,25(OH)2D3 (10 nM) was added to the culture. The effects of Vitamin D on persistence of plasma cells and Ig production over the next 5 days were assessed and were compared to a no Vitamin D controls. Data are the mean ?SEM from triplicate determinates. Significant differences are shown as * (p<0.05). Figure 7. Vitamin D inhibits generation of post-switch memory B cells from naïve B cells. A. Pre-switched naïve B cells (CD19+IgG-CD27-) were sorted from enriched peripheral B cells from three healthy donors. Post-sort analysis of the naïve B cells is shown. The purity of sorted cells was more than 99%. B. Naïve B cells were stimulated as indicated with or without 1,25(OH)2D3 (10 nM) . After 5 days in culture, the B cells were stained for expression of CD19 and IgG or IgM (upper right quadrant). C. The percentages of post-switched memory B cells were compared between culture with or without Vitamin D. D. After 7 days in culture, the number of plasma cells and IgG in the supernatants were analyzed. Data are the mean ? SEM from triplicate determinations of one of three independent experiments. Asterisks indicate the statistical significance between Vitamin D 73 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 treated and control B cells (p?0.01). Figure 8. Vitamin D inhibits XBP1 and ERN1 expression by activated B cells. RNAs were extracted from freshly isolated B cells and B cells cultured with anti-IgM, anti-CD40 and IL-21 with or without 1,25(OH)2D3 (10 nM) for 3 days. The mRNA expression of various gene products was determined by RT-PCR and the fold change was compared to freshly isolated B cells. A. Relative expression of PAX, BCL6, and AID. B. Relative expression of BLIMP1, IRF4, MAT3, XBP1 and ERN1. Data represent the mean ? SEM of three independent experiments conducted in triplicate from different donors. Significant differences between Vitamin D and control responses are shown by * (p<0.05). Figure 9. The effect of Vitamin D on cell cycle regulated genes. RNAs were extracted from freshly isolated B cells and B cells cultured with anti-IgM, anti-CD40 and IL-21 with or without 1,25(OH)2D3 (10nM) for 3 days. The mRNA expression of P27, P21 and P18 was determined by superarray and/or quantitative PCR. A. The relative expression of P21 and P27 determined by Superarray. Data represent the mean? SEM of three independent experiments. B. The relative expression of P21, P27 and P18 determined by quantitative PCR. Data represent the mean? SEM of three independent experiments. Significant differences between Vitamin D treated and control B cells are shown by * (p<0.05). Figure 10. B cells can convert inactive 25(OH)D3 into the active form of Vitamin D. A. CYP27B1 is constitutively expressed by fresh B cells and is induced by stimulation. B cells were analyzed either immediately after isolation or were incubated for 3 days with the indicated stimuli with or without 1,25(OH)2D3 (10nM). The expression of CYP27B1 was determined by RT-PCR relative to B2M mRNA (×106). The means ? SEM of one of three independent experiments are shown . Significant differences between Vitamin D treated and control B cells are shown by * (p<0.05). Significant differences between day 0 and stimulated B cells are shown as ** (p<0.01) B. Purified B cells were stimulated with anti-CD40 and IL-21 with either 10 nM 1,25(OH)2D3 or different concentrations of 25(OH)D3 ranging from 0 to 1000 nM. After 7 days in culture, the cells were washed and 74 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 stained for expression of CD19, CD27 and CD38. The geometric mean fluorescence of CD38 and the frequency of plasma cells (CD27hi/CD38hi) were determined. The culture supernatants were analyzed for IgG production, The 25(OH)D3 dose response (solid line) is compared to 10 nM 1,25(OH)2D3 (dashed line). References: 75 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 附件二 综述 1, 25-二羟维生素D3的免疫学作用及其临床应用 维生素D的主要生理学功能是调节机体的钙磷平衡,近年来随着认识的深入,发 现其调节免疫功能方面新的重要内容,这些新的内容对于我们重新认识其在体内重要 生物学调节作用,拓展其临床应用有极为重要的意义,以下就有关维生素 D与自身免 疫性疾病关系的研究进展进行综述。 1(l , 25( OH) 2D3的生物合成 1, 25-二羟维生素D3(1, 25( OH) 2D3)是维生素D3的活性形式,属于第二甾体类 激素。维生素D3可以从食物中摄取,也可以由皮肤中的7-脱氢胆固醇经紫外线照射异 构而成。1968年,有学者提出维生素D3,本身是没有生物活性的,必须经过代谢激活 才能发挥生物效应[ 1],因此引发了对维生素D活性形式的分离和明确其化学结构的研 究。1968至1970年,维生素D3的活性代谢产物25( OH) D3和1, 25( OH) 2D3才被描述。 研究发现,维生素D3首先经肝细胞线粒体内的羟化酶羟化形成25( OH) D3,这是维生 素D在血液循环中的主要形式,25( OH) D3仍无生物活性,必须经肾小管细胞内羟化酶 作用,最终转化成l , 25( OH) 2D3,才具有生物活性。l , 25( OH) 2D3是体内最重要的维生 素D的活性代谢产物,是主要的Ca++动员激素,在调节机体钙、磷平衡方面起着关键 作用,小肠上皮细胞、甲状腺细胞、肾细胞和骨细胞是其经典的靶细胞。 活性维生素D是通过与存在于细胞内的特异性维生素D受体( Vi t ami n D r ecept or,VDR)结合后发挥生物效应的,其经典生物作用是维持机体的钙、磷平衡, 它的主要活性代谢产物l , 25( OH) 2D3作用于肠、骨、肾等靶器官,调节钙磷水平。?小 肠:l , 25( OH) 2D3通过与存在于小肠上皮细胞内的SVDR结合,从而促进肠上皮细胞对 钙、磷的吸收。?骨:成骨细胞上存在VDR和甲状旁腺激素受体( PmR),在l , 25( OH) 2D3 和Pr H的作用下通过一系列复杂的机制使得骨钙和骨磷动员人血浆中,目前它们联合 作用的机制尚不完全清楚。?肾:l , 25( OH) 2D3与存在于肾远曲小管上皮细胞内的VDR 结合,可以促进肾远曲小管对钙、磷的重吸收。 76 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 2 VDR的结构与功能 维生素D受体( VDR)与其他核内受体具有广泛的同源性,核内受体通常分三个功能 域:N端为可变域,不同受体之间无同源性;中间为DNA结合域(?3D),此域可与靶基 因结合,在不同的受体之间有较高的同源性( 60,,95,),其中包括两个结合DNA所 必需的锌指结构;C端为激素结合域即配体结合域( I 23D),此域在不同受体之间的同 源性为30,,60 ,。 VDR含有427个氨基酸,DBD位于N端1- 114位氨基酸残基,转录活化域位于1- 190位 氨基酸残基,l , 25( OH) 2D3结合域位于115- 427位氨基酸残基,缺失N端1- 104或C端 282/ 373- 427氨基酸片段均对骨钙素基因转录起抑制作用。l , 25( OH) 2D3与VDR- LBD结 合后,VDR- LBD可识别DNA上的调控区并与之结合,该调控区段称激素反应元件,属于 增强子或抑制子,以调控相应基因的表达[ 2]。可以认为,VDR本质上是一个配体依赖 的转录因子,它与l , 25( OH) 2D3一起在调节机体钙、磷代谢,细胞增殖与分化以及免 疫功能等方面起重要作用。VDR可与靶基因启动子区域DNA特异性VDRE结合而调控靶基 因的转录。VDR对靶基因维生素D反应元件( VDRE)的选择和识别有一定的规律,典型 VDRE结构中存在由6bp的保守序列( GCGTGA)组成的重复序列,两个保守序列之间有一 定数目的碱基,称为间隔区碱基,间隔区碱基数目决定受体与VDRE结合的特异性。 VDR的转录调控与众多重要的基因表达有关,包括参与机体钙平衡和骨骼形成的 基因。已经明确VDR能调控骨钙化蛋白、钙结合蛋白、小肠线粒体ATP合成酶、细胞色 素氧化酶亚基等基因的表达。活性Vi t D,与VDR、RXR结合可促进骨钙化蛋白基因转录, 增加骨钙化蛋白的合成。活性Vi t D,可通过VDR调控相关基因转录而控制单克隆骨髓 细胞U937的生长与分化。这些基因包括P21、CD14基因、转录因子HoxA10和Mad1基因 等。另外,VDR可与癌基因c- j un表达产物的同型二聚体或c- j un与c-表达产物形成杂 二聚体A1,在转录方面起拮抗作用,抑制骨钙化蛋白的转录。其他受活性Vi反向调 节的基因有PTH基因、I胶原al基因、ANP基因、GMCSF基因、BSP基因、I FN- r基因和 I L- 2和I L- 12基因等 [ 3] . 3(1, 25( 0H) 2D3在细胞增殖、分化和凋亡中的作用 实验发现, l , 25( OH) 2D3及其类似物在多种培养细胞和癌细胞异种移植的模型中 具有明确的增殖抑制效应。比如,l , 25( OH) 2D3的类似物EB1089在对患HNSCC(头颈部 鳞状细胞癌)的小鼠模型的治疗中能够抑制8O,的肿瘤细胞的生长[ 5]。在乳癌、结 77 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 肠癌和前列腺癌等异种移植模型中也得到相似结果。另外,许多还研究发现, l , 25( OH) 2D3能够诱导多种类型的细胞停留在细胞周期的G期而产生增殖抑制效应。 到目前为止,l , 25( OH) 2D3抑制增殖的机制尚在进一步研究中,可能的机制包括B一转 化生长因子( TGF- B)信号转导的增强、表皮生长因子受体( EGFR)表达的减少、胰岛素 样生长因子( I GF)的成分改变,以及l , 25( OH) 2D3对细胞周期蛋白依赖性激酶( CDK)的 抑制剂p21和p27在转录与转录后水平上的诱导等[ 6]。此外,l , 25( OH) 2D3还能通过抑 制MAPK/ ERK信号转导通路来抑制细胞的增殖 [ 7]、 l , 25( OH) 2D3还能够诱导多种类型细胞的凋亡。比如。人乳腺癌MCF- 7细胞在 100nmol / L的l , 25( OH) 2D3处理48小时后出现凋亡特征:细胞质凝缩、核固缩和染色体 聚集;原位杂交显示出特征性DNA断裂;同时。与凋亡密切相关基因的mRNA和蛋白质 合成水平均明显增加。最近的研究也发现(EB1089能够诱导肝癌细胞和慢性淋巴细胞 性白血病细胞的凋亡。其他实验室以前列腺癌细胞、乳腺癌细胞、结肠癌细胞和卵巢 癌细胞为模型也得到类似结果。到目前为止,l , 25( OH) 2D3诱导细胞凋亡的机制也仍 在进一步探索中(可能的机制有l , 25( OH) 2D3能够抑制Bcl - 2、Akt和I GF的表达;促进 Bax、MEK1和细胞色素C的表达以及增强TNF- a的活性等[ 7]。 4(1,25( 0H) 2D3对免疫细胞的作用 直到1980年,有关维生素D对免疫系统的作用才得到认识[ 10]。l , 25( OH) 2D3的激 素形式对组织和细胞均有影响,但对体液免疫没有直接的影响[ 11]。最近研究表明: 维生素D是一种新的神经内分泌的免疫调节激素,对细胞免疫具有重要的调节作用, 主要表现为对单核,巨噬细胞、T淋巴细胞,树突状细胞以及胸腺细胞增殖分化的影 响和这些细胞功能的影响等 [ 12]。研究发现,VDR可以激活T细胞,以及激活巨噬细 胞合成l , 25( OH) 2D3,对免疫细胞的分化和增殖均有免疫调节的作用[ 11]。 l , 25( OH) 2D3,能抑制原核细胞增殖而间接刺激单核细胞增殖,促使单核细胞向 吞噬作用的巨噬细胞转化,然后将加工处理的病原体传递给辅助T淋巴细胞,增强r干 扰素合成,干扰素又刺激巨噬细胞产生1a羟化酶,形成l , 25( OH) 2D3的正反馈效应。 在单核/巨噬细胞系统中,l , 25( OH) 2D3不仅可使正常外周血单核细胞向巨噬细胞分 化,而且可以加强单核/巨噬细胞的免疫功能[ 13]。并能促进单核/巨噬细胞或调节被 激活的T细胞产生白细胞介素( I L) 1、2、3、6和肿瘤坏死因子( TNF) - a, r。另外,巨噬 细胞本身还具有合成1, 25( OH) 2D3的能力。1, 25( OH) 2D3还能抑制CD4的表达和人体单 78 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 核细胞相关病毒感染[ 14]。l , 25( OH) 2D3缺乏,外周血T淋巴细胞总数及T辅助细胞百 分比明显下降,由于T辅助细胞减少而导致CD4/ CD8比值下降。已知T辅助细胞主要功 能是分泌细胞因子,诱导和增强T、B细胞及巨噬细胞的免疫应答,其数量的减少直接 导致了细胞免疫功能降低,亦使B细胞分化和成熟障碍,导致低免疫球蛋白血症,以 致免疫功能降低[ 15]。 l , 25( OH) 2D3已经被证实作用于T细胞,以及具有调节细胞因子和淋巴细胞抗体 产物的作用。研究表明,l , 25( OH) 2D3的主要作用是抑制表达VDR的T细胞的增殖,而 对静息T细胞无作用[ 11]。这主要依赖于VDR表达的阈值,在某种程度上,通过抑制T 细胞而抑制I L- 2的分泌,通过对T细胞的作用。 l , 25( OH) 2D3也通过T细胞抑制I NF- r的合成,另外,还可以通过刺激I NF- r使巨噬 细胞合成1, 25( OH) 2D3。T细胞的发展代替了具有VDR表达的胸腺髓质细胞,但是不作 用于不成熟的胸腺皮质细胞,目前,在T淋巴细胞发展过程中的精细调节作用仍不完 全清楚,因为在细胞离开胸腺以后,VDR的表达就消失了。近来研究表明,VDR在自然 杀伤NK细胞中存在,指出l , 25( OH) 2D3对病毒及肿瘤引起的感染的免疫反应中具有调 节作用[ 11]。 5(l , 25( OH) 2D3与类风湿性关节炎 慢性炎症性关节疾患中类风湿性关节炎是最常见的,此外还有莱姆关节炎、反 应性关节炎、青年性关节炎等,尽管这些疾病的病因尚不完全明确,但它们都有着一 些相同的病理特征,包括病损局部存在中性粒细胞、巨噬细胞、T淋巴细胞、B淋巴细 胞以及树突状细胞浸润等,这些炎症反应导致了关节囊的增厚、新生血管的形成以及 关节软骨和骨的不可逆性损害。Thl细胞因子I L- 2、TNF- a和I FN- r在发病中起了重要 作用。布氏氏螺旋体( Bor r el i a bur gdor f er i )感染小鼠是制作类风湿性关节炎疾病模 型的方法。 Cant oma等[ 16]发现,在动物模型的饲料中添加l , 25( OH) 2D3 (每天20 ng)可预 防关节炎的发生。认为其机制可能在于活性维生素D抑制了致关节炎性细胞因子如 I L- 2、I NF- a、I FN- r的产生,从而抑制了关节滑膜的增生以及关节软骨和骨的损害。 有研究[ 17]表明在类风湿性关节炎病损部位的关节软骨细胞、巨噬细胞和滑液细胞 上均有 VDR的表达。而且受累关节局部存在维生素D代谢的内分泌调节,局部由巨噬 细胞和滑液纤维母细胞合成的l , 25( OH) 2D3通过调节l a羟化酶、24羟化酶的活性以及 79 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 细胞表面VDR的数目来进行旁分泌控制[ 18]。因此,有作者认为局部l , 25( OH) 2D3的 浓度有可能影响类风湿性关节炎的病程,从而提出直接注射活性维生素 D至受累关节 腔内治疗的可能性。 6. l , 25( OH) 2D3与SLE 有研究认为l , 25( OH) 2D3对SLE患者DCs有免疫调节作用,可能主要作用于髓系树 突状细胞( dendr i t i c cel l s,DCs)。SLE患者的DCs的细胞核上有VDR的表达, l , 25( OH) 2D3对SLE患者髓系DCs作用可能如下[ l 9]:当l , 25( OH) 2D3,与SLE患者DCs 细胞核上的VDR结合后,继而与位于Rel B基因启动子区域的VDREs特异性地结合,从而 抑制Rel B基因的转录活性,使NF- KB蛋白Rel B产生减少,髓系DCs成熟障碍,不能激活 初始反应性T细胞,T- B细胞间的协同刺激作用减弱,由此使致病性的自身抗体产生减 少[ 20],在一定程度上缓解了SLE的病情。对于PDCs( pl as macyt oi d dendr i t i c cel l s, PDCs),l , 25( OH) 2D3的调节作用可能为:由于抑制了髓系DCs的成熟(使作为内源性 I FN诱导因子的自身抗体产生减少(对PDCs的刺激作用减弱,从而使I FN- a的产生减少。 1, 25( OH) 2D3,对SLE患者DCs的免疫调节作用还有待于进一步的研究。 7(l , 25( OH) 2D3与多发性硬化 多发性硬化( mul t i pl e scl er osi s,MS)是一种中枢神经系统的慢性炎症性疾病, 其特征性表现为血管周围有T细胞、巨噬细胞浸润以及神经元细胞的脱髓鞘性改变。 尽管其病因尚不完全清楚,但目前认为它是一种T细胞介导的自身免疫性疾病。实验 性自身免疫性脑脊髓炎( EAE)小鼠在很多方面与人类MS具有相似性, 可以作为MS的动物模型。l , 25( OH) 2D3作为一种免疫调节刺,很多学者应用它来预防 I L~t E鼠发生脑脊髓炎获得了成功。病理检查发现,与对照组相比,治疗组EAE鼠大脑 及脊髓中炎症浸润程度较轻,脱髓鞘区域范围较小。目前关于l , 25( OH) 2D3保护作用 产生的机制及持续时间仍存在不同[ 21- 23]。Mat t er等 [ 21]研究发现, l , 25( OH) 2D3抑制了I L- 12的产生继而影响了I L- 12依赖性Thl细胞的活化,使得致脑脊 髓炎性细胞因子I FN(7的生成减少。Cant oma等[ 22]则认为l , 25( OH) 2D3的保护作用在 于增加了体内转化生长因子B( TGF- B)和I L4等抗炎因子的表达,而不是降低了Th1细胞 因子TNF- a和I FN- r的水平。此外,Mat t er等[ 21]发现短期使用l , 25( OH) 2D3 (从再次 接触抗原后的第5,l 3天,每公斤体重3ug,隔日腹腔注射)即具有长时间的保护作用, 而Cant oma等[ 22]则认为停用l , 25( OH) 2D3治疗会导致脑脊髓炎迅速复发,导致这些 80 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 研究结果差异的原因尚不清楚(但可能与实验中所使用的EAE鼠模型不同有关。与1型 糖尿病相似,近年来也发现维生素D受体基因的序列多态性可能与某些人群MS的易感 性有关 [ 24]。 MS是一种自身免疫性疾病,其治疗主要应用免疫抑制剂,免疫抑制疗法的主要弊 端在于广泛的抑制了机体的免疫功能,而l , 25( OH) 2D3作为一种新型的免疫调节剂, 既能达到预防和治疗自身免疫性疾病的目的,又不引起广泛的免疫抑制作用。一些研 究和来自美国移民局的调查资料显示[ 25],通过提供足够水平的活性维生素D或其类 似物进行早期干预,可能预防遗传易感的个体罹患MS。许多研究也发现,和对照比较, 多数MS病人尤其是女性病人,腰椎和股骨颈骨密度明显减低,而且MS越严重,骨密度 越低,说明维生素D缺乏越严重,而此种缺乏能够通过常规补充安全廉价的维生素D得 到纠正,从而改善MS病人的预后。此外MS病人的经典治疗多使用激素,也容易造成骨 质疏松,骨质丢失,因而使用维生素D预防或治疗MS具有广阔的空间,维生素D治疗的 副作用在于高钙血症,甚至能够造成肾功能损害,而且维生素D的治疗剂量与中毒剂 量间安全范围较小,大量连续应用可以发生中毒。所以需要在治疗过程中摸索其最佳 用量。 9. 1,25( 0H) 2D3与移植排斥 心脏和胰岛移植 Hect or等[26]利用两个MHC不同的小鼠进行心脏移植,观察 l , 25( OH) 2D3对移植排斥的影响。用小剂量的环孢素不能阻止排斥反应的发生,但用 小剂量的l , 25( OH) 2D3( 50 ng/ d)辅以0. 87%的钙离子,能保护小鼠不发生排斥反应, 时问长达100 d。短期使用l , 25( OH) 2D3成功诱导了小鼠对胰岛的同种移植耐受,接受 移植的小鼠从移植第1- 30 d口服小剂量的l , 25( OH) 2D3,( 5ug/ kg,3次/周),未引起 高钙血症。在停药100 d后,仍然检测到小鼠体内以共刺激分子下调和I L- 12分泌显著 减少为特征的DCs。用抗CI M单克隆抗体处理的小鼠能够与移植物相适应,但不产生 移植耐受,所有小鼠在移植后14( 14?2(4) d发生排斥,用l , 25( OH) 2D3和霉酚酸酯( MMF) 同时处理的小鼠中80,长期接受移植物( >70 d),MMF单独应用40,小鼠接受同种胰岛 移植[ 27]。 肾移植 Aschenbr enner等[ 28]报道,在大鼠和小鼠肾移植慢性排斥的模型中, 用l , 25( OH) 2D3显著延长了移植后动物的生存时间,并且减少了肾移植后骨钙的丢失。 l , 25( OH) 2D3抑制了肾移植后TGF- B水平的升高。TGF- B1能够刺激细胞外基质蛋白的合 81 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 成,抑制此蛋白的降解,促进移植器官纤维化;同时,TGF- B1还能刺激平滑肌细胞 迁移至血管内壁,从而使血管内膜细胞增厚、管腔狭窄、造成移植后的慢性排斥和移 植肾存活率下降,应用l , 25( OH) 2D3在体内通过改变VDR和Smad3的水平影响TGF- B1的 水平,从而预防和减少肾纤维化的发生和发展。还有学者利用一种合成的维生素 D类 似物MC1288,研究对大鼠大动脉移植急性和慢性排斥反应的影响。MC1288对VDR的亲 和力与l , 25( OH) 2D3相似,与后者化学结构的差异仅在20位碳原子的立体构型,但是 不易引致高钙血症。结果显示,0(1ug/ kg的剂量仅用了2 d,急性排斥反应中经常发 生的血管外膜的炎症有所改善;MC1288与环孢素联合应用,移植1个月后,组织学观 察证实,血管外膜炎症和内膜增厚均被显著抑制[ 29]。 10(l , 25( OH) 2D3与I型糖尿病 I型糖尿病是一种T细胞介导的器官特异性自身免疫性疾病(VDR基因多态性可能 与该病易感有关,已有学者通过动物实验证实l , 25( OH) 2D3对该病有保护作用,机制 可能在于使具有自身免疫倾向的NOD鼠在免疫调节过程中恢复了抑制性细胞的活性, 促进了致糖尿病性效应细胞的凋亡(在Vi t D缺乏动物口服糖耐量试验中发现糖耐量没 有发生变化,可能是由于糖转运的钠依赖成分减少,影响到肠道对葡萄糖的吸收 [ 30],从而影响糖耐量,降低胰岛素的分泌(在临床上也有许多相关的研究,在一次 欧洲许多地区的实验调查中一致显示:婴儿时期补充Vi t D对I型糖尿病发病有保护作 用,活性Vi t D可以调节免疫,在早期环境暴露的敏感人群中,保护或阻止正在进行 的首发的免疫过程[ 31](Hypponen E[ 32]等的研究发现,补充饮食Vi t D与降低I型糖 尿病发病风险相关,对婴儿确保补充足够Vi t D有助于逆转I型糖尿病发生的增加趋势。 11. l , 25( OH) 2D3与溃疡性结肠炎 溃疡性结肠炎是一种以大肠粘膜和粘膜下层炎症为特点的病因不明的慢性炎症 性疾病(其发生与细胞免疫直接相关(近年研究认为本病很可能为自身免疫性疾病, 大量的细胞因子参与了本病的免疫和炎症过程(在临床研究[ 33]中发现来自溃疡性结 肠炎患者的T淋巴细胞对环孢素A的作用比正常者敏感,用环孢素A和1, 25 ( OH) 2D3 联合治疗可在最低浓度达到抑制T淋巴细胞增殖的作用,从而减少环孢素A的剂量和 毒性( 12. 1,25( 0H) 2D3与恶性肿瘤 阳光照射是机体维生素D的重要来源。流行病学调查显示,机体暴露阳光较多的 82 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 人群(患前列腺癌和结肠癌的概率明显较低。最近(瑞典科学家的把3700名来自瑞典 和丹麦的淋巴瘤患者与3100名健康人进行了比较研究,结果发现那些晒太阳越多越经 常的人,得淋巴瘤的危险越小,尤其是非何杰金氏病[ 4]。这些结果提示,l , 25( OH) 2D3 具有潜在的抗癌效应。实际上许多体外研究表明, l , 25( OH) 2D3在多种人的组织中具有 明确的抗癌作用,包括抗乳癌、前列腺癌、结肠癌、皮肤癌、卵巢癌和白血病等。动 物实验也为l , 25( OH) 2D3的抗癌效应提供了依据。实验发现,l , 25( OH) 2D3及其类似 物任结肠、胃肠道、肝细胞、皮肤和仓鼠颊囊的癌变模型中均发挥了明显的抗癌作用。 l , 25( OH) 2D3的抗肿瘤作用不仅因为抗增生作用,还表现为诱导肿瘤细胞的分化 和成熟。早在1980年代就有报道两种维生素类物质一维甲酸( RA)和维生素D3对人类白 血病细胞系HL- 60有诱导分化作用,对具有原始单核细胞特征的人组织细胞白血病细 胞系U937细胞也有促分化作用[ l 6]。但二者的促分化方向略有不同:RA使HL- 60细胞 向成熟粒细胞分化,l , 25( OH) 2D3使HL- 60细胞只向成熟单核,巨噬细胞分化,二者均 使U937细胞向成熟单核,巨噬细胞分化,使人原始巨核细胞白血病细胞系( HI Meg)细 胞向较成熟巨核细胞分化。l , 25( OH) 2D3的促急性早幼粒细胞白血病NB4细胞和U937细 胞的分化作用是通过细胞核内的VDR产生的,近期研究发现,l , 25( OH) 2D3是通过改 变细胞周期的进程来诱导NB4细胞相单核细胞分化成熟的,表现在增殖能力下降、细 胞形态发生变化和细胞膜表面单核细胞抗原CD,表达增强,而在细胞发生形态和生化 改变之前,细胞周期调节蛋白( cel l cycl e r egul at or y pr ot ei n) Cdk 2、Cdk 1、Cdk 4、RBBP的表达下降,表明细胞周期已经发生了变化。孟凡义等将l , 25( OH) 2D3、全反 式维甲酸和阿糖胞苷联合应用治疗急性早幼粒细胞白血病22例,以HOAP治疗的19 例为对照,结果治疗组CR为100,,达CR时间平均为29(95天,生存期1年以上分别为 84. 2%,均明显高于对照组,且治疗组无严重感染、骨髓抑制等严重并发症。此外, 由于肿瘤的生长及转移结节的形成均需新血管生成的细胞外基质浸入,而 l , 25( OH) 2D3可刺激人的纤维连接蛋白( PN)的合成及抑制鸡胚胎绒毛膜的血管生成, 故认为l , 25( OH) 2D3可抑制肿瘤的转移。 虽然研究显示l , 25( OH) 2D3有抗肿瘤和免疫调节作用,但由l , 25( OH) 2D3引起的高 钙血症以及大剂量时的心脏毒性、肾结石等毒副作用限制了它在治疗中的应用。为了 克服l , 25( OH) 2D3的这些毒副作用,研究转向了l , 25( OH) 2D3的同类物。最近的动物实 验已证实了l , 25( OH) 2D3的同类物19- nor - 1,25( 0H) 2- D2( Par i cal ci t o1)对白血病、 83 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 骨髓瘤和结肠癌细胞具有明显的抗肿瘤活性,同时却无l , 25( OH) 2D3的上述毒性; Par i cal ci t ol通过使细胞周期停滞而抑制急性髓细胞白血病细胞系HL一60、NB4和THP 一1细胞的增殖,诱导骨髓瘤NCI—H929细胞凋亡,并使直结肠癌细胞HT- 29和SW837形 成的肿块缩小;而l , 25( OH) 2D3的另一同类物EB 1089也被发现可诱导慢性B淋巴细胞 白血病细胞的凋亡。 以上是Vi t D在几种较典型而多发的自身免疫性疾病中的作用机理、实验研究和应 用,对于其他一些自身免疫性疾病的作用也在研究和应用中。1,25( OH) 2D3可通过 多种途径来调节机体免疫功能,根据机体的免疫状态,通过增强或抑制免疫功能来发 挥调节免疫状态的作用。l , 25( OH) 2D3作为免疫调节剂用于自身免疫性疾病,不仅可 达到防治效果,还不会有免疫抑制剂造成的副作用(现在部分已由动物实验转向临 床应用(随着Vi t D类似物合成技术的提高,更有效的Vi t D类似物将为免疫性疾病的 治疗提供更广阔的应用前景( 综上所述,l , 25( OH) 2D3是一种新发现的免疫调节剂,即能达到预防和治疗自身 免疫性疾病的目的,又不产生全身性的免疫抑制作用。随着对其保护作用机制以及新 型类似物的研制开发,它在临床上具有广阔的应用前景。 84 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 参考文献 1.Barnett AH(A review of bassl insulins[J](Diabet Med,2003,20(11):873-885( 2. Mangdsdorf DJ,Thummel C,Beato M,et al(The nuclear receptor superfamily:the semnd decade(Cell,1995,83(6):835,83 3. RachezC(Freedrmn LP(Mechnisms; of gene regulation by vitaminD3 receptor,a networrk of CO, activator interactions(Gene,2000。246(1-2):9,2 4. Egan KM et a1(J Natl Cancer lnst,2005(97:161-163 5. Prudeneio J et a1(J Natl Can(yet lnst(2o01(93:745-753 6. Li n R et a1(Endocr i nol ogy(2003(144:749- 753 7. Beer TMet a1(Mol Cancer 7 2004(3:373- 381 8. Li n R et a1(Mol Endoenno1(2002(16:1243- 1256 9. Mar ei nkowska E et a1(Aet a Bi o(?hi m Po1(2002(49:393- 406 10. Del uca HF(Cant ona MT(Vi t ami n D:i t s r ol e and uses i n i mmunol ogy[ J](FASEBJ,2001, 15( 14):2579- 2585 11. Hewi son M(Vi t ami n Dand t he i mune syst em[ J](En—docr i nol,1992,132( 2):173- 175 12.宋亮年(1,25( OH)维生素D,的免疫调节作用[ J](生理科学进展,1994,25:83- 85 13.黄爱萍(维生素D缺乏性佝偻病的免疫功能研究[ J](实用儿科临床杂志,1996,11( 2):67- 69 14.杨月亮,冯学斌,吴福玲,等(反复呼吸道感染患儿免疫状态与血清维生素D关系探讨[ J](实 用医学杂志,1998,14( 1):46. 15.李晓玲(50例维生素D缺乏性佝偻病患儿的免疫检测[ J](福建医药杂志,1998,20( 3):38- 39. 16. Cant or na MT,Hayes CE,Del ueCA HF,et a1([ J](J Nut r,1998,128:68- 72( 17. Tet l ow LC,Smi t h SJ,Mawer,EB, et al([ J](Ann Rheum Di s,1999,58( 2):118- 121( 18. Smi t h SJ(Hayes ME. Sel by PL,et a1([ J](Ann Rheum Di s,1999,58( 6);372- 378( 19. Dong X,Craig T,Xing N,et a1(Direct transcriptional regulation of RelB by 1alpha, 25-dihydroxyvitamin D3 and its analogs:physiologic and therapeutic implications for dendritic cell function(J Biol Chem,2003,278:49378-49385( 20. El st ner E,Kl i nenber g JR, Wal l ace DJ,et al(Vi t ami n D3 and i t s synt het i c an anal ogs i nhi bi t t he spont aneous i n vi t r o i mmunogl obul i n pr oduct i on by SLE- der i ved PBMC(Cl i n l mmunol , 2001,99:82- 93( 21. Mat t er F,Smi mdo S,Gal bi at i F,et a1([ J] _Eur J I mmumol,2OOO,30( 2):498- 508( 22. Cant or na MT,woodwar d WD,Hayes CE,et a1([ J](J I mmunol . 1998,160( 11):5314- 5319( 23. Nashol d FE,Mi l l er DJ,Haye CE([ J](J Neur oi mr nmol,2OOO,103( 2):171- 179( 24. Fukazawa T,Yabe I,Ki l mchi S,et a1([ J] J(J Neur ol Sei,1999,166( 1):47- 52. 25. Hayes CE, Cantorna MT,Deluca HF(Vitamin D and multiple(Proc Soc Exp Bio Med,1997, 216(1):21-27( 26. Hector FD,Margherim TC(Vitamin D:its role and uses in im—munology[J](The FASEB Jouamal, 2001,15(12):2579-2585. 27.Gregori S,Casorati M,Amuchastegui S,et a1( Regulatory T cells induced by 1, 25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance[J](J Immunol,2001,167:1945-1953 28. Aschenbrenner Jk,Hullett DA,Hei~y DM,et a1(1,25-dihydroxyvltamin D3 has salutary efects on renal allografl function [J](Transplant,2000,69:229-23 29. Raisanen-sokolowski AK(Pakkala IS,Samila SP,et a1(A vita(min D analog,MC 1288,inhibits adventitial inflammation and suppresses iutimal lesious in rat aortic allografts[J](Transplanta—tion。 85 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 1997。63(7):936-941( 30. AYESHA I(RAGHURAMULU N(Oral glucose toleran ce is unaltered in vitaminD deficient rat [J](Journal of Nutritional Science and Vitaminolo gy,2000,46 (3):115 31. The EURODIAB Substudy 2 study Group( Vitamin D Supplement in early childhood and risk for Type I (insulin—dependent)diabetes mellitus[J](Dia(betologia (GERMANY), 1999,42 (1):51 32. HYPPONEN E, LAARA E, REUNANEN A, et a1(Intake of vitamin D and risk of type I I diabetes:a birth—cohort study[J](Lancet(England),2001,358 (9292):1500 33. STIO MARIA, TREVES CRISTINA,CELLI ALESSANDERA, et al( Synergistic inhibitory effect of cydosporin A and vitam in D derivatives on T —lymphocyte proliferation in active ulcerative colitis[J](American Journal of Gastroenterology, 2002, 97 (3):679 34.Koeffler HP. Induction of human acute myelogenous leukemia cells:therapeutic implications. Blood,1983;62:709. 35.Olsson IL,Breitman TR. Induction of differentiation of the human histiocytic lymphoma cell line U-937 by retinoic acid and cyclic adenosine 3’:5’ –monophosphate- inducting agents. Cancer Res,1982;42:3924. 36.Tanaka H, Abe T, M,iyaura C, et al. 1-alpha, 25-dihydroxycitamin D3 induces differentiation of human promyelocytic leukemia cells (HL-60) into monocyte – macrophages, but not into granulocytes. Biochen Biophys Res Commun. 1983;117:86. 37.Bhatia M, Kirklad JB, Kelly A. Monocytic differentiation of acute promyelocytic leukemia cells in response to 1,25(OH)2-D3 is independent of nuclear receptor binding. J Biol Chem, 1995;270(27):15926. 38.Ward JO, Mcconnell MJ , Carlile GW ,et al . The acute promyelocytic leukemia-associated protein, promuelocytic leukemia zinc finger , regulates 1,25-dihydroxycitamin D(3)-induced monocytic differentiation of U937 cells through a physical interaction with vitamin D3 receptor. Blood,2001;98(12):3290. 39.程涛,严舫,万景华。维甲酸与l,25( OH) 2D3对白血病细胞的联合促分化效应。上海医学,1991; 14(5):280。 3 40.程涛,严舫。l,25( OH) 2D联合小剂量化疗对白血病细胞的体外作用。第二军医大学学报, 1991;12(5):425。 41.孟凡义,刘海川,楼方定,等。l,25( OH) 2D全反式维甲酸和阿糖胞苷联合治疗急性早幼粒细 3 胞白血病。临床内科杂志,1995;12(3):35. 3 42.余怀勤,孙旦澄,许湘贤,等。l,25( OH) 2D治疗急单白血病获完全缓解1例。现代诊断与治 疗,1995;(5):312. 43.Studzinski GP , Moore DC. Sunlight-can it precent as well as cause cancer? Cancer Res , 1995;55:4014. 44.Mellibovsky L , Diez A, J, et al. Long-standing remission after 25-OH D3 treament in a case of chronic myelomonocytic leukaemia. Br J Haematol,1993;85:811. 45.Kumagai T, O’Kelly J Said JW, et all. Vitmain D3 analog 19-nor-1,25-dihydroxyvitamin D2 :antitumor activity against leukemia,myeloma, and colon cancer cells. J Natl Cancer Inst,2003;95(12):896. 46.Molnar I,Kute T,Willigham MC,et al .19-nor-1 alhpa,25-dihydroxyvitamin D2 (PARICALCITOL):effects on clonal proliferation,differentiation,and apoptosis in human leukemic cells.J Cancer Res Clin Oncol,2003:129(1):35 47.Pepper C,Thomas A,Hoy T,et al.The vitamin D3 analog,EB1089,induces apoptosis via a 86 上海交通大学 ,, ,,,,,,,,,, , , , , , , , , 附件 p53_independent mechanism involving p38 MAP kinase activation and suppression of ERK activity in B-cell chronic lymphocytic leukemia cells in vitro Blood,2003:107(7):2454. 48. Mehta RG,Mehta RR.Vitamin D and cancer.J Nutr Biochem,2002;13(5):252. 49. Lathers DM,Clark JI,Achile NJ,et al.Phase 1Bstudy to immune responses in head and neck cancer patients using escalating doses of 25-hydroxyvitamin D3. cancer Immunother,2004;53(5):422. 50.Clark CS,Konyer JE,Meckling KA.1 alpha,25-di-hydroxyvitamin D3 and bryostatin-1 synergize to induce monocytic differentiation of NB4 acute promyelocytic leukemia cells by modulating cell cycle progression.Exp Cell Res,2004;294(1):301. 51. Danilenko M,Wang Q,Wang X,et al. Carnosic acid potentiates the antioxidant and prodifferentiation effects of I alpha,25-dihydroxyvitamin D3 in leukemia cells but does not promote elevation of basal levels of intracellular calcium.Cancer Res,2003;63(6):1325. 87 上海交通大学 ,, , ,谢 , , , , , , , , ,,,,,,,,,, 致 谢 临近毕业,心中感慨万千,回望来时路程,感谢所有一路陪我走来的老师、同事, 同学以及家人。 首先要感谢求知路上给我指引、循循以诱的长辈。感谢我的导师顾越英教授,她 对我的关心和爱护是从生活到学习无所不在的,那种如沐春风的温暖感觉令我感受至 深。她开朗宽容的处事态度,临难不惧,勇挑责任的治病风格是我为人为医追求的境 界。感谢陈顺乐教授,他高瞻远瞩、胸襟开阔的大家风范,令我无比景仰,他出色的 国际外交才能,以及对后辈成长的关心使我受益匪浅。正是有了他的引荐,我才得以 有机会遇到 Dr. Peter Lipsky,作为我在美期间的导师,把我领入了细胞免疫基础研究 的领域,他的博学、对科研前沿动态敏锐的捕捉能力、以及严谨的治学态度使我受益 良多。正是他们引领我初窥了风湿免疫学的科学殿堂,并像航标一样给我正确的方向。 其次,要感谢这一路上始终给我支持和帮助的科室前辈和同事。感谢我所在的风 湿免疫科以及科室的每一位成员,是他们营造了和谐宽松的学习和工作环境。鲍春德 教授、王元教授、杨程德教授、和沈南教授给我的支持和鼓励;感谢叶萍老师、叶霜 师兄、陈晓翔师兄、戴岷师兄、王晓栋和李挺在实验中给予的鼎力援助,使我度过了 不少难关。另外,也一并对关心帮助我科室同事们表示衷心的感谢。他们对事业的执 著追求和积极向上的精神使我对我们科室的前景充满信心。 课题研究期间,我有幸得到美国国立卫生研究院的资助前往完成基础研究部分。 这一特殊的经历让我收获颇多。在这里,我得到了 Autoimmune Branch/NIHMS的所 用同事的关心和帮助,感谢 Gar y Si ms, Randy Fi scher和 Rachel Et t i nger等在实 验技术上给予的指导,感谢 Ji mSi mone和 Raf ael Vi l l as mi l在细胞分选中给予的协 助。这段独立生活的经历,同时让我锻炼了能力和意志,让我受益终身。在此期间, 我结识了许多朋友,如尹洪恩、吴传庆、关明、王婷等,共聚的时间虽然不长,亦师 亦友般的真挚友情却会铭记一生。也让我们在今后的人生道路上互勉互助,一起向前。 感谢科研部何幼琴老师、孔宪明部长的帮助,感谢研究生处陈小明处长、高老师、 杜老师的帮助。 最后由衷地感谢始终在我身后支持我的全家,尤其是我出国 2年,我父母的无私 付出,丈夫的充分信任以及儿子唐成源小朋友给我带来的无限快乐使我得以顺利完成 88 上海交通大学 ,, , ,谢 , , , , , , , , ,,,,,,,,,, 学业。作为母亲在儿子牙牙学语、蹒跚学步的时候却无法陪伴在侧,深感愧疚,也许 这是我一辈子无法弥补的缺失,但相信,有付出才会有收获。 经过的路,有苦有乐,然而正是有了导师的指引,同道的相助,朋友的鼓励和家 人的相伴,一路的风景才会如此显得美丽。也正是有了这些,今后的路才会愈见宽阔。 环顾四周,只想由衷地说声:“谢谢~” 89 上海交通大学 ,, ,,,,,,,, ,,,,,,,,, ,,,,,,,,,, 已刊文章 第一作者文章: 1. Sheng Chen, Yue-Ying Gu, Chun-De Bao, et al. An 18-year follow-up study of a lupus cohort in Shanghai. APLAR Journal of Rheumatology. 2006,9(4):327-1. 2. Sheng Chen, Gary P. Sims, Xiao Xiang. Chen,et al. Modulatory Effects of 1,25-Dihydroxyvitamin D3 on Human Primary B Cells.(submitted) 其它文章: 1. Chuan-ging Wu, Anuradha Budhu, Sheng Chen, et al. Effect of Hepatitis C Virus Core Protein on the Molecular Profiling of Human B Lymphocytes Mol Med. 2006 Jan–Mar; 12(1-3): 47–53. 2. 参编书籍 1.陈顺乐主编的《红斑狼疮》中的“抗组蛋白抗体”章节。 90
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