【作者】 樊晓光；

【导师】 杨林花；

【作者基本信息】 山西医科大学 ， 内科血液免疫学， 2006， 博士

【摘要】 目的实验性免疫性甲状腺炎是自身免疫性疾病研究的一个典型实例。为了开创一个研究的新途经,阐明T、B细胞在实验性免疫性甲状腺炎中的免疫作用及DC递呈Tg产生的T细胞克隆的免疫作用,阐明实验性免疫性甲状腺炎的诱发机制,我们以甲状腺球蛋白(Thyroglobulin,Tg)为抗原,树突状细胞为抗原递呈细胞建立了抗原特异性的T细胞克隆,并进行了被动转移实验。材料及方法NOD.H-2h4鼠饲养于SPF无菌环境中,喂养0.05%碘化钠8周后,每两周采血,用ELISA测Tg水平,然后每只小鼠用100ug Tg的剂量混匀于福氏完全佐剂中免疫小鼠两次,用淋巴细胞增殖实验优化Tg的含量,寻找树突状细胞摄取递呈Tg的最适抗原量,然后,将免疫后NOD.H-2h4小鼠的脾细胞作为T细胞与同基因鼠的并与抗原孵育过的树突状细胞共培养。同时选用这六个T细胞克隆中的四个T细胞克隆---F11,2D11,H8,G8,将NOD.H-2h4鼠分为X射线照射组和正常NOD.H-2h4鼠组,进行了T细胞克隆的被动转移实验。结果建立了六个Tg特异性的T细胞克隆,这些细胞克隆在体外培养时间大于两年以上,流式细胞仪检测其表型均为CD4+,而且分泌IFN-γ,不分泌IL-4。T细胞克隆被动转移实验表明:所有的四个T细胞克隆均不能引起正常NOD.H-2h4鼠的甲状腺炎。四个克隆中三个T细胞克隆可引起X线照射组NOD.H-2h4鼠的甲状腺炎,而小鼠的照射剂量(475拉德)足够去除受鼠的抑制性细胞的功能。2D11克隆被动转移的结果亦表明:T细胞克隆在体内有辅助B细胞产生抗体的能力。结论抗原特异性的T细胞克隆的建立产生及维持,开创了一个研究自身免疫性疾病的新途经。树突状细胞起抗原递呈细胞的作用,在T细胞系及T细胞克隆的建立中是极为重要的,否则,T细胞即使在Tg及IL-2存在的培养环境中也仅能存活数天。T细胞克隆被动转移的实验,除了可以了解病理产生过程,转移的T、B细胞存活的时间长短外,也有助于了解T、B细胞的免疫调节作用及自身免疫性甲状腺炎的诱发机制,为研究实验性免疫性甲状腺炎的病理产生过程提供了理论依据和实验手段。更多还原

【Abstract】 ObjectiveA case in point is experimental autoimmune thyroiditis, which is the example of autoimmune diseases. The ability to generate, maintain, and use cloned lines of T cells reactive for self-antigens has opened up a new avenue of investigation for researchers. To elucidate the immune function of T cell and B cell and T cell cloning, which were stimulated by Tg (thyroglobulin, Tg) presenting dendritic cells (DCs) in experimental autoimmune thyroiditis from bone marrow of NOD.H-2 h4 mice and to elucidate the mechanism of induction in experimental autoimmune thyroiditis(EAT), Antigen specific T cell clones have been investigated by dendritic cell as the antigen-presenting cells primed with thyroglobulin(Tg).Materials and MethodsNOD.H-2 h4 (Non-obese-Diabetes) mice under SPF(specific-pathogen free) condition were given water with 0.05% iodine for 8 weeks and mice were bled biweekly intervals to measure their level of Tg by ELISA then some of them were immunized Tg 100ug per mouse in complete Freund adjuvant twice. Before sacrificed those immunized mice, proliferation assay has been done to optimize the amount of Tg for the dendritic cells to uptake in order the antigen-presenting cells to present Tg to T cells. Finally, splenocytes taken from Tg immunized mice were co-cultured with dendritic cells from bone marrow of syngeneic mice, which were cultured, irradiated and incubated with Tg as the feeder cells. We also did passive transfer experiment to NOD.H-2 h4 mice by using T cell clones. That is, four out of six T cell clones, F11, 2D11, H8, G8, were transferred to either group of normal NOD.H-2 h4 mice or group of irradiated NOD.H-2 h4 mice.ResultsSix Tg-specific T cell clones were established and they were cultured in vitro over two years and all of them proliferated to Tg and their phenotype are CD4+ by FACS analysis as well as secreted IFN-γinstead of IL-4.Passive transfer experiment by T cell clones indicated four T cell clones were not caused thyroiditis in the normal NOD.H-2 h4 mice and three out of four clones can induce thyroiditis in irradiated NOD.H-2 h4 mice and irradiated dose (475 RAD) to the NOD.H-2 h4 mice is sufficient to remove the function of suppressive cells in receipt mice those have the function to help B cell to produce antibody.ConclusionsThe ability to generate, maintain, and use cloned lines of T cells reactive for self-antigens has opened up a new avenue of investigation for researchers. Dendritic cells, which act as Tg更多还原