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脐血论文:脐血 树突状细胞 裸鼠 肿瘤

2017-11-25 6页 doc 25KB 17阅读

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脐血论文:脐血 树突状细胞 裸鼠 肿瘤脐血论文:脐血 树突状细胞 裸鼠 肿瘤 脐血论文:脐血 树突状细胞 裸鼠 肿瘤 【中文摘要】和近年来,随着人口老龄化及人类生存环境的恶化,恶性肿瘤的发病率和病死率均呈现明显升高的趋势。目前临床肿瘤的治疗普遍还是采用手术、放疗和化疗等传统方法。但这些方法难以清除体内的微小肿瘤病灶且对机体损伤较大,在杀伤癌细胞的同时,往往也伴随着正常组织细胞的损伤。随着人们对肿瘤发生发展机制的进一步认识和肿瘤免疫分子生物学和生物工程技术的进展,肿瘤生物治疗迅速发展,成为肿瘤治疗的第四种治疗模式。树突状细胞(dendritic cells, D...
脐血论文:脐血 树突状细胞 裸鼠 肿瘤
脐血论文:脐血 树突状细胞 裸鼠 肿瘤 脐血论文:脐血 树突状细胞 裸鼠 肿瘤 【中文摘要】和近年来,随着人口老龄化及人类生存环境的恶化,恶性肿瘤的发病率和病死率均呈现明显升高的趋势。目前临床肿瘤的治疗普遍还是采用手术、放疗和化疗等传统方法。但这些方法难以清除体内的微小肿瘤病灶且对机体损伤较大,在杀伤癌细胞的同时,往往也伴随着正常组织细胞的损伤。随着人们对肿瘤发生发展机制的进一步认识和肿瘤免疫分子生物学和生物技术的进展,肿瘤生物治疗迅速发展,成为肿瘤治疗的第四种治疗模式。树突状细胞(dendritic cells, DCs)因具有抗原呈递能力和活化初始T细胞的能力而成为肿瘤免疫治疗的研究热点。但DCs在肿瘤患者外周血和组织中含量较少,体外扩增也较难达到理想的治疗用剂量。因此,探讨从健康产妇脐血分离诱生DCs的方法,研究其体内外的抗肿瘤效应及机制,有助于为脐血来源的DCs及细胞毒性T细胞的临床应用打下基础。方法采集健康足月分娩产妇脐血50-80ml,分离出人脐血单个核细胞(Human Umbilicus blood mononuclear cell, HUBMC),将培养瓶静置2小时,取贴壁生长细胞,在含15%胎牛血清1640培养液中加入GM-CSF、IL-4和SCF,培养诱生人脐血树突状细胞(Human Umbilicus blood dendritic cell, HUBDC)。取对数生长期的BGC823肿瘤细胞,用反复冻融法取得肿瘤细胞提取物作为肿瘤抗原,负载HUBDCs,促使HUBDCs成熟。于普通光学显微镜下观察HE染色后的HUBDCs形态,并用流式细胞仪对其进行表型鉴定。将HUBDCs中的非贴壁细胞采用尼 龙毛柱法分离出T细胞并鉴定其纯度。观测负载抗原成熟的HUBDCs对T细胞的活化增殖能力(混合淋巴细胞反应)。采用MTT比色法检测活化的T细胞对BGC823靶细胞的杀伤。动物实验部分,首先建立荷瘤动物模型,将BGC823细胞注射于裸鼠并观察成瘤情况,记录肿瘤大小。将荷瘤裸鼠随机分成四组,治疗如下:A组:体外负载抗原的HUBDCs和未激活T细胞;B组:体外经HUBDCs诱导活化的特异性T细胞(CTL); C组:负载BGC823抗原的HUBDCs; D组:未经HUBDCs致敏的新分离T细胞。观察裸鼠体内肿瘤的生长情况并分别记录。结果用细胞因子GM-CSF、SCF和IL-4配伍能诱导人脐血贴壁单个核细胞向DC分化,加入肿瘤抗原提取物能促使HUBDCs成熟,HE染色光镜下观察HUBDCs形态符合典型毛刺状、树枝状DCs形态,流式分析仪检测细胞表型:MHC-?、MHC-?、CD86(协同刺激分子)、CD54(黏附分子)、CD11c均较诱导前有显著升高。其中,CD86表达率为40.59%?3.27%,CD54为59.21%?6.32%,CD11c表达率为67.01%?5.17%,MHC-?和MHC-?分别为:42.37%?10.11%和56.31%?6.76%,与诱导前相比,差异具有统计学意义P<0.01。同种混合淋巴细胞反应显示:HUBDCs体外可以使抗原特异性T细胞活化增殖为效应T细胞(cytotoxic T lymphocytes, CTL),且CTL对靶细胞BGC823可以产生特异性的抑制及杀伤。在动物实验中,成功建立人胃癌BGC823荷瘤裸鼠模型。按实验方案分别对各组治疗后,注射负载抗原HUBDCs +新分离T细胞的A组和注射特异性CTL的B组都有明显抑瘤效应,至30天时A组肿瘤大小为:211mm3B组为:153mm3,B组抑瘤效应更加显著;C组单独注射 负载抗原的HUBDCs, D组注射未致敏新分离的T细胞均无明显抑瘤效 果,至30天时C组,D组肿瘤大小分别为:1093 mm3和1022mm3。C组 D组肿瘤细胞仍快速生长。结论1)利用细胞因子能从人脐血单个核细 胞(HUBMC)中诱导出具有典型形态、表型和具有活化T细胞功能的 HUBDCs;2)成熟人脐血DC与T细胞共同移植,能显著增强荷瘤裸鼠的 抗肿瘤能力。 【英文摘要】Background and objectIn recent years, with the population aging and environmental deterioration, the incidence and mortality of malignant tumor continue to show an upward tendency. In clinical practice, surgery, radiotherapy and chemotherapy is the traditional method of treatment of cancer. However, these methods are difficult to eliminate small focuses inside the body, and moreover, cause great damage to human body. The malignant tumor was wiped out, at the expense of the normal tissues and cells of our body were damaged. With further understanding of the mechanism of tumor and developing of oncomolecularbiology and biotechnology, cancer biotherapy is developing rapidly. It has become the forth method for cancer treatment. Dendritic cells have the unparalleled ability which can present antigen, active and proliferate the naive T cell (Tn). With the unique features, DCs have become the hot research. However, DCs in the peripheral blood and tissues are lower. It is difficult to achieve therapeutic dose in vitro, even though amplification mrthod. In this paper, we try to find a new way that separating and deducing DCs from umbilicus blood which come from healthy maternity. Study anti-tumor effect and mechanism of DCs in vivo.Lay the foundation for clinical application of DCs and CTL.MethodsCollect the umbilicus blood 50-80ml from healthy term delivery maternity then separate human umbilicus blood mononuclear cell by centrifugation. Place the culture flask at rest for 2 hours then collect adherent cells from the bottom. The adherent cells was cultured in fetal calf serum culture fluid with the presence of multiple cytokines (GM-CSF,IL-4,SCF) to generate HUBDCs. Tumor antigen BCG823 was prepared from the exponential phase of growth tumor cells thorough freeze-thawing method. After HUBDCs were pulsed with tumor antigen, morphology dyed by HE was observed under optical microscope and the phenotypes was analyzed by flow cytometry. T cells were separated by nylon fiber from non-adherent cells and identify the purity. The activation and proliferation ability of active T lymphocyte was analyzed through Mixed lymphocyte reaction (MLR). Detect the killing power of active T lymphocyte by methyl thiazolyl tetrazolium (MTT) colorimetry. For in vivo experiment, animal models were built:transplant BGC823 cells to nude mice. Recode the status of tumor-bearing nude mice and the growth of tumor.Divide tumor-bearing mice into 4 groups in accordance with the random principle. Treatment of each group are as follow:A group:antigen-pulsed HUBDCs and inactive T lymphocyte, B group: antigen specific CTLs; C group:HUBDCs pulsed with tumor antigen; D group: inactive T lymphocyte which separated freshly from umbilicus blood. Tumor growth of each group was recorded respectively.ResultHUBDCs can be induced from HUBMC with the presence of cytokines (GM-CSF, IL-4, SCF). Tumor antigens were added to mature HUBDCs. The Morphology of HUBDCs dyed by HE conformed with typical morphology features of DCs:dendritic and extensively branched. The phenotypes were analyzed by flow cytometry. Compared with before induction, tumor antigen pulsed HUBDCs expressed high-level phenotypes, including:CD86(co-stimulatory molecules),CD80, CD54(adhesion molecule),MHC-?and?.The positive percentage of CD86 was 40.59%?3.27%, CD54 was 59.21%?6.32% CD11c was 67.01%?5.17% MHC-?and MHC-?were:42.37%?10.11% and 56.31%?6.76%. The difference was statistically significant compared with before induction. The result of Mixed lymphocytes reaction (MLR) shows that:HUBDCs can stimulate and proliferate antigen-specific T lymphocyte into cytotoxic T lymphocyte (CTL).In vitro; CTL can inhibit and kill the BGC823 target cells. In zoopery part, Both A group which injected with tumor antigen pulsed HUBDCs and allogeneic inactive T lymphocyte and B group which injected with cytotoxic T lymphocyte have obvious antitumor effect. At 30 days after, A group of tumor size was 211mm3, B group was:153 mm3, B Group is more obvious effect. C group which inject with tumor antigen pulsed HUBDCs and D group inject with inactive T lymphocyte have no effect. To 30 days, C and D group of tumor size was 1093 mm3 and 1022 mm3.ConclusionHUBDCs that have morphology and phenotypes feature can be induced successfully from HUBMCs, and they have the capacities of stimulating native T cell activation, proliferation and differentiation into antigen specific CTLs.Transplantation of antigen pulsed HUBDCs and allogeneic T lymphocyte can enhance the anti-tumor ability in tumors bearing nude mice. 【关键词】脐血 树突状细胞 裸鼠 肿瘤 【英文关键词】umbilicus blood dendritic cell nude mice tumor 【目录】人脐血来源树突状细胞诱导CTL对BGC823肿瘤细胞的 抑制作用 摘要 4-7 Abstract 7-9 引言 12-15 材料和方法 15-22 结果 22-28 讨论 28-34 结论 34-35 参考文献 35-37 综述部分 树突状细胞抗肿瘤作 用研究进展 37-52 参考文献 49-52 附录部分 52-53 个人简历 52-53 致谢 53 【索购全文找】1.3.9.9.3.8.8.4.8 1.3.8.1.1.3.7.2.1 同时提供论文写作一对一辅导和论文发表服务。 【说明】本文仅为中国学术文献总库合作提供,无涉版权。作者如有异议请与总库或学校联系。
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