宿主免疫应答对日本血吸虫生长、发育、生殖及对虫卵肉芽肿形成的影响研究（可编辑）

宿主免疫应答对日本血吸虫生长、发育、生殖及对虫卵肉芽肿形成的影响研究（可编辑） 华中科技大学 博士学位论文 宿主免疫应答对日本血吸虫生长、发育、生殖及对虫卵肉芽肿 形成的影响 姓名:程喻力 申请学位级别:博士 专业:病原生物学 指导教师:李雍龙 20090501 华 中 科 技 大 学 博 士 学 位 论 文 宿主免疫应答对日本血吸虫生长、发育、生殖及虫卵肉芽肿形成的影响 华中科技大学同济医学院病原生物学系 博士研究生 程喻力 导师 李雍龙 中文摘要 传统观点认为寄生虫与宿主间的相互关系表现为寄生虫对宿主的损害和宿主对 寄生虫的抵抗，而宿主对寄生虫的抵抗主要表现为宿主的免疫应答可以杀灭宿主体内 的部分寄生虫并限制寄生虫的再感染，与此同时，寄生虫则利用各种方式逃避宿主的 免疫攻击。 然而，新近研究及流行病学资料显示，在宿主免疫功能低下时，血吸虫的发育或 生殖力受限，提示宿主的免疫反应对血吸虫的影响可能不光局限于限制或杀灭，血吸 虫对宿主的免疫攻击可能也不仅限于逃避，我们的假设是:在寄生虫与宿主长期共进 化过程中，血吸虫为了生存，不仅设法逃避宿主的免疫攻击，而且还可能利用宿主免 疫反应提供的某些效应分子(如细胞因子、抗体等)，促进自身的发育或生殖，即宿 主的免疫应答可能有助于血吸虫的发育或生殖。但是，迄今为止，哪种类型的免疫应 答或免疫反应中哪些因素及效应分子有利于血吸虫的生长和繁殖，还远不清楚。 研制有效的抗血吸虫疫苗是防治血吸虫病的根本途径，疫苗的研究工作已超过半 个世纪，然而，迄今为止，该研究仍无实质性进展。由于目前对血吸虫感染免疫的认 识是建立在传统观点之上的，而抗血吸虫疫苗研究的理论基础也是建立在免疫反应能 杀灭虫体或限制其产卵能力之上的，完全没有考虑诱导的免疫应答是否会对血吸虫产 生有利作用。因此，深入认识血吸虫感染的免疫对血吸虫生长、发育及生殖的影响对 疫苗研究尤为重要, 阐明影响血吸虫发育和繁殖的免疫应答类型或效应分子，将为在 3 华 中 科 技 大 学 博 士 学 位 论 文 血吸虫疫苗研究中如何激活不利于血吸虫发育的因素而避免激活有利于血吸虫发育1 的因素提供理论基础。 本研究通过观察日本血吸虫在不同免疫缺陷动物体内的发育、生殖及宿主肝脏虫 卵肉芽肿的病理特点，探讨不同免疫因素对日本血吸虫发育、生殖及宿主虫卵肉芽肿 形成的影响。 本论文包括如下三部分: 第一部分 第二部分 第三部分 T 细胞对日本血吸虫生长、发育、生殖及对虫卵肉芽肿形成的影响 信号转导与转录活化因子 Stat4 和 Stat6 对血吸虫生长、发育、生殖及对虫 卵肉芽肿形成的影响 宿主细胞因子 IL-12、IL-4 对血吸虫生长、发育、生殖及对虫卵肉芽肿形 成的影响 第一部分 T 细胞对日本血吸虫生长、发育、生殖及对虫卵肉芽肿形成的影响 裸鼠是由于染色体内裸体位点的等位基因(Nu 基因)发生纯合而形成的突变小鼠。 该小鼠由于先天性胸腺缺陷，导致其体内不能产生胸腺依赖淋巴细胞(T 细胞)及其 诱导的各类免疫反应。我们利用裸鼠此特性，研究宿主 T 细胞缺陷对日本血吸虫生长、 发育、生殖及对宿主肝脏虫卵肉芽肿形成的影响。 材料与方法 正常 BALB/c 小鼠及相同遗传背景的裸鼠每只均感染日本血吸虫尾蚴 25 条。两 组小鼠各分 3 小组，分别于感染后第 28 天、35 天及 42 天剖杀小鼠，经门静脉灌注法 收集虫体，计数总虫荷，然后于解剖显微镜下分别计数雌、雄虫荷，并观察 虫体发育 情况。所有虫体经 10%福尔马林固定后，盐酸卡红酒精染色，并置于载玻片上用中性 基金项目:国家自然科学基金项目(No.30471511) 4 华 中 科 技 大 学 博 士 学 位 论 文 树胶封固。将封固虫体的玻片置于光学显微镜下等倍数码成像，并导入 Image pro 5.0 图像分析程序，测量并计算虫体的长度。镜下观察虫体，计数雌虫子宫内虫卵数。 门静脉灌注收集虫体后摘取小鼠肝脏，从肝右叶切取部分新鲜肝组织，置于 10% 福尔马林液中固定后，石蜡包埋、切片，HE 染色，显微镜下组织病理学观察。选择 切片中含单个虫卵的肉芽肿，利用 Image pro 5.0 图像分析系统，以虫卵内毛蚴为中心， 测量肉芽肿直径。剩余肝组织经 5%KOH 溶液消化后，显微镜下计数虫卵，并计算肝 组织中每对虫体平均产卵数。 所有数据均以???s 表示，用 SPSS12.0 进行统计学处理，两组间比较采用 Student’s 双尾 t 检验。 结 果 实验结果显示，两组小鼠体内总虫荷及雌雄虫配对数在感染后第 28、35 及 42 天 均无差异，但感染后第 28 天，裸鼠体内雌虫及雄虫长度均明显低于正常 BALB/c 小 鼠，且差异具极显著性(p 0.01)。然而，至感染后第 35 天，该差异缩小至无显著性 p 0.05 。至感染后第 42 天，两组小鼠体内虫体长度已趋于一致。 裸鼠肝组织中每对虫体平均产卵数在感染后各时间点均显著低于正常 BALB/c 小 鼠(p 0.05)。裸鼠体内雌虫子宫虫卵数在感染后第 28、35 及 42 天均略低于 BALB/c 小鼠，但差异均无显著性(p 0.05)。BALB/c 小鼠在感染后第 28 天雌虫子宫内均有 虫卵产生，而裸鼠在感染后第 28 天有部分雌虫(28.5%)子宫内未见虫卵，至感染后 第 35 天仍有个别雌虫(4.7%)子宫内不含虫卵，直至感染后第 42 天，所有被检测雌 虫子宫内才均含有虫卵。 裸鼠肝脏虫卵肉芽肿平均直径明显小于 BALB/c 小鼠，且差异具极显著性 (p 0.01)感染后第 42 天，正常 BALB/c 小鼠肝脏可见典型虫卵肉芽肿形 成，肉芽肿 周围未见明显肝细胞坏死。裸鼠肝脏内肉芽肿较不典型，且浸润细胞显著减少，其中， 以嗜酸性粒细胞减少最为明显。另外，裸鼠肝脏虫卵肉芽肿周围可见大面积肝细胞坏 死。 5 华 中 科 技 大 学 博 士 学 位 论 文 结 论 上述结果表明，宿主 T 细胞缺陷并不影响日本血吸虫在宿主体内的生存，且日本 血吸虫仍可在宿主体内发育成熟并产卵。然而，T 细胞缺陷时，虫体发育及产卵明显 延迟，提示 T 细胞介导的免疫反应及某些相关特异性免疫因子的产生可能有利于血吸 虫在宿主体内的移行或发育，但通过动态观察，首次发现宿主 T 细胞缺陷对日本血吸 虫发育及产卵的影响是一过性的，主要作用于发育早期。 另外，虫卵肉芽肿的形成有赖于 T 细胞，T 细胞对介导肉芽肿内嗜酸性粒细胞的 浸润具有重要作用。 关键词:日本血吸虫;T 细胞缺陷;虫卵肉芽肿 第二部分 信号转导与转录活化因子 Stat4 和 Stat6 对血吸虫生长、发育、生殖及 对虫卵肉芽肿形成的影响 近年来，大量研究报道感染血吸虫的宿主体内辅助性 CD4+ 辅助性 T 细胞 Th cell 的分化方向，即向 Th1 细胞或 Th2 细胞亚群的分化，对宿主的保护性免疫效应及 虫卵肉芽肿的免疫病理反应具有重要影响。 Stat4 和 stat6 分别为启动 Th 细胞向 Th1 细胞和 Th2 细胞亚群分化的重要信号转 导分子。当 Stat4 缺乏时，刺激 Th 细胞向 Th1 细胞分化的信号途径被阻断，导致 Th1 型免疫反应缺陷，而 stat6 缺乏则导致 Th2 型免疫反应缺陷。 本研究利用 Stat4 基因敲除(Stat4-/-)小鼠和 Stat6 基因敲除(Stat6-/-)小鼠，分 别观察宿主 Th1 型免疫缺陷和 Th2 型免疫缺陷对日本血吸虫发育生殖及宿主虫卵肉芽 肿形成的影响。 6 华 中 科 技 大 学 博 士 学 位 论 文 材料与方法 正常 BALB/c J 小鼠及相同遗传背景的 Stat4-/-、Stat6-/-小鼠每只均感染日本血吸虫 尾蚴 25 条。三组小鼠各分 3 小组，分别于感染后第 28 天、35 天及 42 天剖杀小鼠， 经门静脉灌注法收集虫体，计数总虫荷及雌、雄虫荷，并观察虫体发育情况。所有虫 体卡红染色后，测量虫体的长度，计数雌虫子宫内虫卵数。门静脉灌注收集虫体后摘 取小鼠肝脏，从肝右叶切取部分新鲜肝组织，置于 10%福尔马林液中固定后，石蜡包 埋、切片，HE 染色，显微镜下病理组织学观察，测量切片中含单个虫卵的肉芽肿直 径。剩余肝组织经 5%KOH 溶液消化后，显微镜下计数虫卵，并计算肝组织中每对虫 体平均产卵数(检测方法同第一部分)。 所有数据均以???s 表示，用 SPSS12.0 进行统计学处理，多组间比较采用单因素 方差分析 ANOVA 。 结 果 实验结果显示，Stat4-/-和 Stat6-/-小鼠体内总虫荷及雌雄虫配对数在感染后第 28、 35 及 42 天与 BALB/c J 小鼠相比均无差异。三组小鼠体内虫体长度在感染后各时间点 亦均无差异(p 0.05)。 Stat4-/-和 Stat6-/-小鼠日本血吸虫感染后第 28 天，肝组织中每对虫体平均产卵数与 BALB/c J 小鼠无差异，至感染后第 35 天，Stat4-/-和 Stat6-/-小鼠肝组织中产卵数略高 于 BALB/c J 小鼠，至感染后第 42 天，该差异进一步增大但始终无显著性(p 0.05)。 各组雌虫子宫内虫卵数在感染后第 28 天无明显差异，至感染后第 42 天，Stat4-/-和 Stat6-/-小鼠雌虫子宫内虫卵数略高于 BALB/c J 小鼠，但差异无显著性(p 0.05)。该 结果与各组小鼠肝组织内每对虫体产卵量的变化趋势一致。 此外，Stat4-/-小鼠肝脏虫卵肉芽肿纤维化较对照组组更为明显，而 Stat6-/-小鼠肝 脏虫卵肉芽肿较不典型，纤维化不明显。肉芽肿直径在各组间无明显差异(p 0.05)。 7 华 中 科 技 大 学 博 士 学 位 论 文 结 论 本研究发现 Stat4-/-和 Stat6-/-小鼠体内虫荷、虫体长度、肝组织中每对虫体平均产 卵数及雌虫子宫内虫卵数与正常小鼠均无明显差异，表明单纯 Th1 型或 Th2 型免疫应 答对日本血吸虫的生存、发育及生殖均无明显影响。Stat4 缺陷促进虫卵肉芽肿纤维化， 提示 Th1 型免疫应答对虫卵肉芽肿的纤维化有抑制作用。Stat6 缺陷不利于虫卵肉芽 肿的形成及纤维化，提示 Th2 型免疫应答对肉芽肿的形成及纤维化起关键作用。 关键词:日本血吸虫;Stat4;Stat6;虫卵肉芽肿 第三部分 宿主细胞因子 IL-12、IL-4 对血吸虫生长、发育、生殖及对虫卵肉芽肿 形成的影响 研究报道，细胞因子的刺激对Th细胞的分化具有重要作用。初始CD4+Th细胞的 分化受IL-12和IL-4的双向调节。IL-12可促使初始Th细胞向Th1细胞分化，而IL-4可促 使Th细胞向Th2细胞分化。血吸虫感染中有关细胞因子的调控作用是目前的研究热点。 关于细胞因子功能的研究传统方法是通过静脉注射抗细胞因子单克隆抗体来实 现其中和作用。然而，观察血吸虫在终宿主体内发育及虫卵肉芽肿的形成需经历一个 较长的过程，而抗体在宿主体内会逐步降解而不能持续发挥中和作用，将能分泌相应 单克隆抗体的杂交瘤细胞直接接种于实验动物体内，则可保证单克隆抗体的持续分泌 并有效的分布于血液及组织中。 本实验将抗 IL-12 和抗 IL-4 的杂交瘤细胞分别接种于小鼠背部皮下，通过杂交瘤 细胞持续分泌的相应单克隆抗体中和小鼠体内的 IL-12 或 IL-4，观察这些细胞因子缺 陷对血吸虫生长、发育、生殖和对虫卵肉芽肿形成的影响。 8 华 中 科 技 大 学 博 士 学 位 论 文 材料与方法 将分泌抗小鼠 IL-12 的杂交瘤细胞 C 17.8 和分泌抗小鼠 IL-4 的杂交瘤细胞 11B11 经 PBS 混悬后定量注射于 BALB/c 小鼠背部皮下，共接种两次，两次之间间隔三周。 小鼠分为如下 3 组: 1)抗 IL-12 组(小鼠接种抗 IL-12 杂交瘤细胞) 2)抗 IL-4 组(小鼠接种抗 IL-4 杂交瘤细胞) 3)对照组(小鼠注射等体积 PBS) 每只小鼠于首次接种杂交瘤细胞后 4,5 天感染日本血吸虫尾蚴 25 条。于感染后 第 0、28 及 42 天用双抗夹心 ELISA 法检测各组小鼠血清中 IL-12 和 IL-4 水平。于感 染后第 28 天和第 42 天，剖杀小鼠收集虫体，计数总虫荷及雌雄虫配对数，对比各组 小鼠体内虫体发育生殖状况及肝脏虫卵肉芽肿病理改变。所有虫体经盐酸卡红酒精染 色，数码成像，并导入 Image pro 5.0 图像分析程序，测量并计算虫体的长度，以了解 虫体发育情况;显微镜下计数雌虫子宫内虫卵数;门静脉灌注收集虫体后摘 取小鼠肝 脏，经消化后，计算肝组织中每对虫体平均产卵数;感染后第 42 天，观察小鼠肝组 织病理学变化。 所有数据均以???s 表示，用 SPSS12.0 进行统计学处理，多组间比较采用单因素 方差分析 ANOVA 。 结 果 接种抗 IL-12 或抗 IL-4 杂交瘤细胞的小鼠血清中均未检测到相应细胞因子。三 组间虫荷在感染后第 28 天及 42 天均无显著性差异。然而，在感染后第 28 天，除了 抗 IL-12 组外，抗 IL-4 组和对照组均可见少量幼虫。抗 IL-12 组体内虫体长度在感染 后 28 天及 42 天均大于抗 IL-4 组和对照组，且差异具有显著性(p 0.05)。而抗 IL-4 组和对照组之间虫体长度无明显差异。 抗 IL-12 组小鼠肝组织中每对虫体平均产卵数在感染后第 28 天显著高于抗 IL-4 组及对照组(p 0.05)，然而至感染后第 42 天，该差异缩小且无显著性。 9 华 中 科 技 大 学 博 士 学 位 论 文 抗 IL-12 组雌虫子宫内虫卵数在感染后第 28 天明显高于对照组(p 0.05)。抗 IL-12 组在感染后第 28 天所有雌虫子宫内均已含有虫卵，而抗 IL-4 组和对照组在该时间点 均有少量雌虫(抗 IL-4 组:6.3%;对照组:14.3%)子宫内未见虫卵。 在感染后第 42 天，抗 IL-12 组小鼠肝脏虫卵肉芽肿直径明显大于对照组(p 0.05)， 而抗 IL-4 组虫卵肉芽肿较不典型，且直径略小于对照组，但差异无显著性(p 0.05)。 与对照组相比，抗 IL-12 组肉芽肿纤维化明显增强，而抗 IL-4 组较不明显。 结 论 利用杂交瘤细胞皮下接种的方法能持续有效的中和小鼠血清中相应细胞因子。 IL-12 或 IL-4 缺陷小鼠体内虫荷与正常小鼠无显著性差异，提示细胞因子 IL-12 或 IL-4 对宿主体内血吸虫的存活无明显影响。然而，IL-12 缺陷有利于血吸虫发育，提示 IL-12 对血吸虫发育可能有阻碍作用。IL-4 对日本血吸虫的发育无明显作用。抗 IL-12 组产 卵提前且感染早期产卵量较正常组高，然而随着感染时间的延长，其产卵量与正常小 鼠无明显差异，提示 IL-12 对雌虫生殖的抑制作用可能是通过抑制其发育而实现的， 此抑制作用是一过性的。抗 IL-4 处理可减弱虫卵诱导的肝组织纤维化，而抗 IL-12 处 理则明显增强肝脏的纤维化，提示 IL-4 可促进肝脏虫卵肉芽肿的纤维化，而 IL-12 则 对纤维化有抑制作用。 关键词:日本血吸虫;白介素 12;白介素 4;虫卵肉芽肿 全文小结 综上 3 部分所述，本研究结论如下: 1.宿主 T 细胞缺陷时，日本血吸虫发育及产卵明显延迟，提示 T 细胞介导的免疫反应 及某些相关特异性免疫因子的产生可能有利于血吸虫在宿主体内的移行或发育，且 T 细胞对虫卵肉芽肿的形成及肉芽肿内嗜酸性粒细胞的浸润具有重要作用。 通过动态观察，首次发现宿主 T 细胞缺陷对日本血吸虫发育及产卵的影响是一过 10 华 中 科 技 大 学 博 士 学 位 论 文 性的，主要作用于发育早期。 2. 利用 Stat4-/-和 Stat6-/-小鼠作为动物模型，首次证明单纯 Th1 型或 Th2 免疫应答对 日本血吸虫生长、发育或生殖均无明显影响。Stat4 缺陷促进虫卵肉芽肿纤维化的形成， 提示 Th1 型免疫应答对虫卵肉芽肿的纤维化有抑制作用;Stat6 缺陷不利于虫卵肉芽 肿的形成，提示 Th2 型免疫应答对肉芽肿的形成及局限化起关键作用。 3.通过将杂交瘤细胞直接接种于小鼠皮下，以借助其持续分泌的单克隆抗体中和相关 细胞因子，观察 IL-12 或 IL-4 缺陷对日本血吸虫发育、生殖及虫卵肉芽肿形成的影响。 首次证实 IL-12 对血吸虫的发育、生殖及对肉芽肿的纤维化有抑制作用，其中对血吸 虫的发育及生殖具的抑制作用是一过性的，而 IL-4 对日本血吸虫的发育无明显影响， 但可促进肝脏虫卵肉芽肿的纤维化。 综合上述结果，我们认为宿主免疫应答类型及某些细胞因子与虫卵肉芽肿的发 生、发展密切相关，而能影响血吸虫在宿主体内生长、发育或生殖的是某些 细胞因子; 鉴于单纯 Th1 型或 Th2 免疫应答对日本血吸虫生长、发育或生殖均无明显影响，因此 我们推测细胞因子对血吸虫生长、发育或生殖的影响可能不是通过单纯 T 细胞应答类 型而发挥作用的，细胞因子对血吸虫生长、发育或生殖影响的具体作用机制还有待进 一步研究。 11 华 中 科 技 大 学 博 士 学 位 论 文 The effects of specific immune deficiency on the development of worms and granuloma formation in mice infected with Schistosoma japonicum Department of Pathogenic Biology, Tongji Medical College, Huazhong university of Science and Technology Doctor candidate Cheng Yu-li Supervisor Li Yong-long Abstract Traditional opinion concerning the relationship between hosts and parasites is that parasites damage their hosts and meanwhile the hosts bring up resistance against the invading parasites through activation of immune response. However, some new study evidences have showed that the growth and oviposition of schistosome was impeded in immune suppressed or immune deficient host. It suggests that the effect of host immune responses to schistosome infection is not only resistance and the reaction of schistosome to host immune attack is more than escape. It's our assumption that during the long period of co-evolution of schistosome with its host, it is possible that schistosome has developed adaptation to the inner environment of its host and even receives some host-derived immune signals to complete its own development. However, it is still not clear that which type of immune response or immune factors might facilitate the growth and development of the invading parasites. So far the rationale of research on schistosome vaccine is based rather narrowly on the traditional opinion that host’s immune response to schistosome infection is exclusively against the development of the parasite. However, this rationale may ignore the possibility that some immune factors in host might be benefit to the parasite. Thus, renewing the immumological opinion on schistosome infection is much important. The purpose of this study is to explore the impact of a deficient immune system on the 12 华 中 科 技 大 学 博 士 学 位 论 文 development of schistosoma japonicum and granuloma formation. We observe the growth and egg-laying of S.japonicum in a mouse model of specific immune deficiency, and accordingly clarify the effects of those immune factors on the development of worms and granuloma formation. The main contents of this thesis include: Part one. The effects of T cell on the development of worms and granuloma formation in mice infected with Schistosoma japonicum Part two. The effects of Signal transducers and activators of transcription Stat -4 and Stat-6 on the development of worms and granuloma formation in mice infected Schistosoma japonicum Part three. The effects of cytokines Interleukin IL C12 and IL-4 on the development of worms and granuloma formation in mice infected Schistosoma japonicum Part one The effects of T-cell deficiency on the development of worms and granuloma formation in mice infected with Schistosoma japonicum Nude mice have a mutated homozygous recessive Nu gene and lack a thymus which is essential for the production of T cells. These mice are unable to mount most types of immune responses, which allowed us to explore the impact of a T cell-deficient immune system on the development of the parasites and the granuloma formation. Materials and methods Intact BALB/c mice and nude mice in BALB/c background were infected with 25 S.japonicum cercariae. The growth and development of the parasites and host tissue pathological change were compared between two groups 28, 35 and 42 days post infection. Worms were collected from the hepatic portal system by perfusion with physiologic saline. 13 华 中 科 技 大 学 博 士 学 位 论 文 The total number of worms and the mean number of worm pairs were counted under a dissecting microscope. After fixed by 10%formalin, all worms were stained by carmine and observed under a compound microscope. Digital images of each worm were obtained using a digital camera. Parasite length was measured from the digital images using “imagepro 5.0”software. Female worms were examined under the microscope and the eggs in the gravid uteri were counted. Infected mice at day 42 post infection were evaluated for liver granuloma formation induced by eggs. Liver was removed and the right lobe was fixed in 10% buffered formaldehyde, embedded in paraffin and cut into sections. The sections were stained with haematoxylin and eosin HE . Representative HE-stained liver sections from each animal were scanned under microscope. For every granuloma containing a single egg, the granuloma size was determined by the measurement of the diameter at the mid-transmiracidial level of the egg. The remaining part of the liver was digested in 5% KOH for counting the total number of eggs per liver and calculating the liver eggs per worm pair. Results were expressed as mean?standard deviation ???s . Student’s two-tailed t-test was used to determine the differences between the experimental and control groups. Results There were no significant differences in worm burden between BALB/c mice and nude mice 28，35 and 42 days post infection. The mean parasite length of either female or male worms in nude mice was found significantly shorter than that in normal BALB/c mice 28 days after infection p 0.01 . This difference lessened 35 days post infection p 0.05 . And the mean parasite length for both groups was found close to each other 42 days post infection. The number of eggs per worm pair in the liver of nude mice remained significantly less than that in BALB/c mice 28, 35 and 42 days post infection p 0.05 . The number of 14 华 中 科 技 大 学 博 士 学 位 论 文 uterine eggs of ovigerous females in nude mice was always slightly lower than in BALB/c mice but, none of the differences was significant p 0.05 . All female worms collected from BALB/c mice at 28, 35 and 42 days after infection contained eggs in their uteri. By contrast, in nude mice, a few female worms had no eggs at 28 days 28.5% or 35 days 4.7% post infection. At day 42, histological examination showed that the granuloma size in nude mice was significantly smaller than that in BALB/c mice p 0.01 . The cellular infiltration within gralunomas in nude mice was much less than that in normal BALB/c mice. A significantly selective diminution in the number of eosinophils within the granulomas was noted in nude mice. In addition, a large area of hepatocytes necrosis in vicinity of eggs was observed in nude mice. But this hepatocytes necrosis was minimal in normal BALB/c mice. Conclusion This study indicates that the survival of S.japonicum within the host would not be significantly affected by T cell deficiency of nude mice. Maturation could still be reached. But, maturation and oviposition of the parasites could be delayed in nude mice. It suggests that T cell induced immune response and related immune factors may be benefit for the migration or development of the worms. Through the dynamic observation, we found the effects on the development of worms was temporary and especially exerted on the early stagy of infection. The deficiency of T cell dependent immune mechanisms could impair granuloma formation, especially the eosinophilia infiltration in the granuloma which is associated with infection by S. japonicum. Key words: Schistosoma japonicum; T-cell deficiency; granuloma 15 华 中 科 技 大 学 博 士 学 位 论 文 Part two The effects of Signal transducers and activators of transcription Stat -4 and Stat-6 on the development of worms and granuloma formation in mice infected Schistosoma japonicum Recent studies indicated that the differentiation of CD4+T cells in S. japonicum infected mice, to Th1 cells or Th2 cells is crucially important for host protective immune response and eggs induced granuloma immunopathology. In this part, we studied the development and fecundity of S. japonicum in Stat4-/- mice and Stat6-/- mice beginning 4 weeks after infection and accordingly explore the effects of Th1-type immune deficiency and Th2-type immune deficiency on the development of S. japonicum and the granuloma formation respectively. Materials and methods Intact BALB/c J mice and Stat4-/-, Stat6-/- mice in BALB/c background were infected with 25 S japonicum cercariae. The growth and development of the parasites and host tissue pathological change were compared between two groups 28, 35 and 42 days post infection. Worms were collected from the hepatic portal system by perfusion with physiologic saline. The total number of worms and the mean number of worm pairs were counted. All worms were stained by carmine and parasite length was measured from the digital images. Female worms were examined under the microscope and the eggs in the gravid uteri were counted. Infected mice at day 42 post infection were evaluated for liver granuloma formation induced by eggs. Liver was removed and the right lobe was fixed in 10% buffered formaldehyde, embedded in paraffin and cut into sections. The sections were stained with haematoxylin and eosin HE . Representative HE-stained liver sections from each animal were scanned under microscope. For every granuloma containing a single egg, the granuloma size was determined. The remaining part of the liver was digested in 5% KOH 16 华 中 科 技 大 学 博 士 学 位 论 文 for counting the total number of eggs per liver and calculating the liver eggs per worm pair. Results were expressed as mean?standard deviation ?? ? s and analyzed by SPSS12.0. The data were analyzed using one-way analysis of variance ANOVA . Results No significant difference was found in the total number of worms and number of worm pairs among Stat4-/-, Stat6-/- and BALB/c J mice at 28, 35 and 42 days post infection p 0.05 . The parasite length among the three groups was also not significantly different p 0.05 . The number of eggs per pair of worms in the liver of the three groups was not significantly different at 28 days post infection. However, this number in the livers of Stat4-/- and Stat6-/- mice was slightly higher than that in BALB/c J mice but still not statistically significant at 42 days post infection p 0.05 . The tendency of the number of uterine eggs of ovigerous females in three groups was similar to the results of the liver eggs at all time points. The fibrosis around eggs in the liver was increased in Stat4-/- mice. The liver granulomas in Stat6-/- mice were not well formed. No significant difference in granuloma size around the single eggs in the liver was found among the three groups p 0.05 . Conclusion The results indicate that the Th1 or Th2 immune deficiency has no conspicuous effect on the survival, development and fecundity of S. japonicum. Th1 immune deficiency facilitated the egg induced granuloma firosis. This suggests that Th1 immune response could inhibit the liver fribrosis which is induced by eggs. The deficiency of Th2 immune response could impair granuloma formation. It suggests that Th2 immune response play an important role for the granuloma formation. Key words: schistosoma japonicum; Stat4; Stat6; granuloma 17 华 中 科 技 大 学 博 士 学 位 论 文 Part three The effects of cytokines Interleukin IL C12 and/or IL-4 deficiency on the development of worms and granuloma formation in mice infected Schistosoma japonicum Cytokines present during the primary stimulation were found to have an important influence on naive Th cell differentiation. Differentiation of na?ve CD4+ Th cell is reciprocally regulated by IL-12 and IL-4. IL-12 was shown to directly promote the development of Th1 cells, whereas IL-4 was found essential for the development of Th2 cells. A great deal of research effort on schistosomiasis has also focused on the effects of these cytokines on schistosome infection. Intravenous injection of anti-cytokine monoclonal antibody is traditionally used for functional study of cytokines. Such an approach may not guarantee all the IL-4 and IL-12 in the blood and tissue are blocked neutralized by the monoclonal antibodies, since schistosomes experience a long duration of migration and development and in vivo antibody degradation occurs over time. Direct inoculation of the animals with hybridoma cell promises sustained antibody secretion which could be sufficient to block the effect of cytokines on the development of schistosome. In the present study, we injected anti-mouse IL-12 C 17.8 and anti-mouse IL-4 11B11 hybridoma cells subcutaneously into the upper backs of mice to block the function IL-12 and IL-4 in vivo to observe the effects of IL-12 and/or IL-4 deficiency on the development of S. japonicum. Materials and methods The identified Hybridoma cells producing anti-mouse IL-12 C 17.8 and/or anti-mouse IL-4 11B11 suspended in PBS was injected subcutaneously into the upper backs of BALB/c mice twice at a three week interval. 18 华 中 科 技 大 学 博 士 学 位 论 文 Mice were divided into 3 experimental groups as follows: 1 Anti-IL-12 2 Anti-IL-4 mice injected with anti-IL-12 Hybridoma cells mice injected with anti-IL-4 Hybridoma cells 3 Intact control (mice injected with PBS) Each mouse was infected with 25 S. japonicum cercariae 4 to 5 days after the first injection of hybridoma cells. The growth and development of the parasites and host tissue pathological change were compared among four groups 28 and 42 days post infection. IL-4 and IL-12 concentrations in serum were measured at 0, 28 and 42 days post infection by ELISA. Worms were collected and worm number was counted under a dissecting microscope. All worms were stained by carmine and parasite length was measured from the digital images using “imagepro 5.0”software. Female worms were examined under the microscope and the eggs in the gravid uteri were counted. Livers were removed for calculation of the liver eggs per worm pair. Infected mice at day 42 post infection were evaluated for liver granuloma formation induced by eggs. Results were expressed as mean?standard deviation ?? ? s and analyzed by SPSS12.0. The data were analyzed using one-way analysis of variance ANOVA . Results IL-12 and IL-4 were not detected in sera of the related hybridoma cell inoculated mice. No significant difference was found in the number of worms among the three groups at 28 and 42 days post infection. However, larva worms were found in all the groups except anti-IL-12 group at 28 days post infection. Worms recovered from mice with anti-IL-12 were of increased length compared with those from other groups at both 28 and 42 days post infection p 0.05 . No significant difference was found in the length of both female and male worms between anti-IL-4 treated mice and intact control. The number of eggs per pair of worms in the liver of anti-IL-12 treated mice was significantly higher than that in anti-IL-4 treated mice and intact control mice at 28 days 19 华 中 科 技 大 学 博 士 学 位 论 文 post infection p 0.05 . But this difference was lessened and not statistically significant at 42 days post infection. The number of uterine eggs of ovigerous females in anti-IL-12 treated mice was significantly higher than that in intact control mice at 28 days post infection p 0.05 . All female worms collected from anti-IL-12 treated mice at 28 days post infection contained eggs in their uteri. In comparison, a few female worms which bore no eggs were found in other two groups at day 28. The granuloma size around the eggs in the liver in anti-IL-12 group was significantly increased p 0.05 but slightly diminished in anti-IL-4 treated group p 0.05 compared to that in control group. The liver granulomas in single anti-IL-4 treated mice were not well formed. The fibrosis around eggs in the liver was dramatically increased in mice treated with anti-IL-12 but was decreased in anti-IL-4 treated group. Conclusion Inoculating the hybridoma cells subcutaneously into the upper backs of mice could efficiently neutralize the related cytokines. Neutralization of IL-12 or IL-4 appeared not to influence the survival of S. japonicum. However, IL-12 may play an impeditive role in the growth of S. japonicum. IL-4 exerts no special effect on the growth of S. japonicum in host. Oviposition of mature worm pairs in anti-IL-12 was initially hastened at early stage of infection but not further enhanced with the time increasing. Anti-IL-4 treatment could decrease egg-induced hepatic fibrosis. Neutralization of IL-12 could promote the granuloma formation and fibrosis. Key words: schistosoma japonicum; IL-12; IL-4; granuloma Summary According to the three parts of study，the summary is as follows: 1. In this study, maturation and oviposition of the S. japonicum could be delayed in nude 20 华 中 科 技 大 学 博 士 学 位 论 文 mice. It suggests that T cell induced immune response and related immune factors may be benefit for the migration or development of the worms. In addition, T cell may play an important in granuloma formation, especially the eosinophilia infiltration in the granuloma which is associated with infection by S. japonicum. Through the dynamic observation, we conclude the effect of T cell deficiency on the development of worms is temporary and especially exerts on the early stagy of infection. 2. By studying the infection of S.japonicum in Stat4-/- and Stat6-/- mice, we confirm that the Th1 or Th2 immune deficiency has no conspicuous effect on the survival, development and fecundity of worms. Stat4 deficiency facilitated the egg induced granuloma firosis. This suggests that Th1 immune response could inhibit the liver fribrosis which is induced by eggs. The deficiency of Stat6 could impair granuloma formation. It suggests that Th2 immune response play an important role for the granuloma formation. 3. We injected anti-mouse-IL-12 and anti-mouse-IL-4 hybridoma cells subcutaneously into the upper backs of mice to block the function of IL-12 and IL-4 in vivo to observe the effects of IL-12 and IL-4 deficiency on the development of S. japonicum. Neutralization of IL-12 or IL-4 appeared not to influence the survival of S.japonicum. However, IL-12 may play an impeditive role in the development of S.japonicum but this effect is only temporary. IL-12 could inhibit the egg-induced hepatic fibrosis. IL-4 exerts no special effect on the development of S.japonicum in host but could promote granuloma formation and hepatic fibrosis. In conclusion, from this study we hypothesize that the type of Th cell induced immune response and related cytokines are associated with granuloma formation and fibrosis. The development and oviposition of S. japonicum may much more depends on the level of some immune factors such as cytokines involved in schistosome infection but not essentially associates to a high polarization of Th1 or Th2 type response. However, the mechanism on how cytokines exert effects on the parasite development is not yet clear. 21 华 中 科 技 大 学 博 士 学 位 论 文 主要英文缩略词 ANOVA BSA CD CTL / Tc DTH ECF EGF ELISA EPG ESP FCS HE HIV HRP ICAM-1 IFN-α IFN-γ IL analysis of variance bovine serum albumin cluster of differentiation cytotoxic T cell delayed type hypersensitivity eosinophil chemotactic factor Precurosor epidermal growth factor enzyme linked immunosorbent assay eggs per gram eosinophil stimulation promoter fetal calf serum hematoxylin-eosin human immunodeficiency virus horseradish peroxidase intercellular adhesion molecule-1 interferon-alpha interferon-gamma interleukin 1 方差分析 牛血清白蛋白 分化群 细胞毒性 T 细胞 迟发型超敏反应 嗜酸性粒细胞趋化因子 表皮生长因子 酶联免疫吸附测定 每克含虫卵数 嗜酸性粒细胞刺激促动因子 胎牛血清 苏木素-伊红 人类免疫缺陷病毒 辣根过氧化物酶 细胞间粘附分子-1 α 干扰素 γ 干扰素 白介素 华 中 科 技 大 学 博 士 学 位 论 文 MIP-1α NK cell OD OPD PBS PDGF RAG rpm SCID SEA SPF macrophage inflammatory protein-1α natural killer cell optical density O-phenylenediamine phosphate buffered solution platelet-derived growth factor recombination activating gene Rounds per minute severe combined immunodeficiency disease soluble egg antigen specific-pathogen free 巨噬细胞炎症蛋白-1α 自然杀伤 细胞 光密度 邻苯二胺 磷酸盐缓冲溶液 血小板源性生长因子 重组活化基因 转/分 重度联合免疫缺陷病 可溶性虫卵抗原 无特殊病原体 Stat Signal transducers and activators of 信号转导与转录活化因子 transcription Th1 Th2 TGF-β TNF-α Type 1 T helper cell Type 2 T helper cell transforming growth factor -beta tumor necrosis factor-alpha 2 1 型辅助性 T 细胞 2 型辅助性 T 细胞 转化生长因子-β 肿瘤坏死因子-α 独创性声明 本人郑重声明，本学位论文是本人在导师指导下进行的研究工作及取得的研究成 果的总结。尽我所知，除文中已经标明引用的内容外，本论文不包含任何其他个人或 集体已经发表或撰写过的研究成果。对本文的研究做出贡献的个人和集体，均已在文 中以明确方式标明。本人完全意识到本人将承担本声明引起的一切法律后果。 学位论文作者签名: 日期: 年 月 日 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，即:学校有权保 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 人授权华中科技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: 日期: 年 月 日 日期: 年 月 日 华 中 科 技 大 学 博 士 学 位 论 文 第一部分 T 细胞对日本血吸虫生长、发育、生殖及对虫卵肉芽肿形成的影响 前 言 传统观点认为寄生虫与宿主间的相互关系表现为寄生虫对宿主的损害和宿主对 寄生虫的抵抗，而宿主对寄生虫的抵抗主要表现为宿主的免疫反应可以杀灭体内的部 分寄生虫并限制寄生虫的再感染，与此同时，寄生虫则利用各种方式逃避宿主的免疫 攻击。 然而，新近研究及流行病学资料显示，在感染血吸虫的 T 细胞缺陷小鼠，虫体发 育迟缓且产卵量减低[1];经可的松处理的小鼠感染血吸虫后，虫荷数较正常小鼠低[2]; 经放射处理过的小鼠，其体内血吸虫产卵量降低[3];预先经尾蚴糖萼免疫过的小鼠其 体内虫荷高于正常小鼠[4];血吸虫雌虫产卵量依赖 IgE 的存在等[5]。提示宿主的某些 特异性免疫反应可能会对血吸虫的生长、发育或生殖能力产生影响。新的流行病学资 料显示，合并感染 HIV 的曼氏血吸虫病患者，其每克粪便虫卵数(E