NOD鼠甲状腺炎动物模型T细胞克隆的建立

目的为了阐明实验性免疫性甲状腺炎的诱发机制,本研究以甲状腺球蛋白(thyroglobulin,Tg)为抗原,树突状细胞(dendritic cells,DCs)为抗原提呈细胞(antigen-presenting cells,APCs),建立了T细胞克隆。方法NOD鼠饲养于无菌环境中喂养0.05%碘化钠8周后,每2周采血,用酶联免疫吸附试验(ELISA)测Tg水平,然后每只小鼠用100μg Tg的剂量混于完全弗氏佐剂(complete Freund adjuvant,CFA)中免疫小鼠2次,用增殖实验优化Tg的含量,寻找DCs摄取提呈Tg的最适抗原量,然后将免疫后鼠的脾细胞作为T细胞与同基因鼠的与抗原孵育过的DCs培养。结果建立了3个Tg特异性的T细胞克隆,这些细胞克隆在体外培养时间>2年以上,流式细胞仪检测其表型均为CD4+,而且分泌干扰素(IFN)-γ,不分泌白细胞介素(IL)-4。结论DCs起APCs的作用,在T细胞系及T细胞克隆的建立中起极为重要的作用。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,此实验结论将为研究实验性免疫性甲状腺炎的诱发机制提供理论依据和实验手段。

Establishment of T cell clone in NOD mice model of thyroiditis

Objective To elucidate the mechanism of experimental autoimmune thyroiditis(EAT) induction, T cell clones have been established with dendritic cell as the antigen-presenting cells primed by thyroglobulin(Tg). Methods NOD-H-2h4(non-obesity-diabetes)mice under SPF(specific-pathogen free) condition were given water with 0.05% NaI for 8 weeks and mice were bled biweekly intervals to measure their levels of Tg by ELISA, then some of them were twice immunized by Tg 100 μg per mouse in complete Freund adjuvant. Before those immunized mice sacrificed, proliferation assay had been done to optimize the amount of Tg for the dendritic cell 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 feed cells. Results Three 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 was CD4+ by FACS analysis as well as secreted IFN-γ instead of IL-4. Conclusion Dendritic cells, which act as Tg presenting cells, were critical and important for the survival of T cell lines and T cell clones, otherwise T cells can only maintain several days although free-Tg and IL-2 existed in the medium, which will provide a procedure to research on mechanism and induction of EAT.