雷公藤内酯醇对自身反应性T细胞的作用机制研究

Effect of triptolide on autoreactive T cells

通过体外实验观察雷公藤内酯醇(Triptolide,Tri)对自身反应性T细胞的作用,为以后将Tri用于体内治疗EAE的研究建立基础。采用CCK-8法检测不同浓度Tri对小鼠成纤维细胞株L929生长的影响;采用MOG35-55肽段免疫C57BL/6小鼠建立EAE疾病模型,获取MOG35-55特异性T细胞,3H掺入法检测Tri对其增殖的影响;ELISA方法检测培养上清中IL-17、IFNγ-、TNFα-、IL-4、TGF-β的含量;流式细胞术检测Tri对Th1和Th17细胞分化的影响。结果Tri浓度≤30nmol/L时,对L929细胞生长未见明显细胞毒性;Tri呈剂量依赖性地抑制MOG35-55特异性T细胞增殖,抑制炎症细胞因子IL-17、IFN-γ、TNF-α的表达,并增加IL-4、TGF-β的表达水平,同时抑制na ve T细胞向致病性Th1和Th17细胞的分化。结果表明,Tri可通过抑制MOG35-55特异性T细胞的增殖,改变MOG35-55特异性T细胞分泌细胞因子的格局及抑制Th1和Th17细胞分化的途径影响自身反应性T细胞,从而为Tri体内治疗EAE的研究提供依据,也为临床研究治疗MS药物提供实验基础。

To observe the effect of triptolide（Tri） on autoreactive T cells in vitro so as to lay a foundation for the treatment of EAE in vivo,effect of different concentrations of Tri on the proliferation of fibroblast cell line L929 was detected by CCK-8 method and the EAE mouse model was established by immunization of mice with MOG35-55 peptide.The proliferation of MOG-specific T cells was investigated by 3H-TdR incorporation method,and ELISA assay was used to detect the secretion of IL-17,IFN-γ,TNF-α,IL-4 and TGF-β in the culture supernatants.In addition,the effect of Tri on the Th1 and Th17 cell differentiation was detected by flow cytometry.The experimental results showed that there was no statistically significant cytotoxicity on the L929 cells when the concentration of Tri was lower below 30 nmol/L.Tri could inhibit the proliferation of MOG-specific T cell in a dose-dependence manner.Also,it could down-regulate the expression of inflammatory cytokines,including IL-17,IFN-γ and TNF-α,up-regulate the expression of anti-inflammatory cytokine IL-4 and TGF-β and inhibit the differentiation of Th1 and Th17 cells.It is evident that Tri may affect the autoreactive T cells through various mechanisms,such as inhibition of proliferation of MOG35-55-specific T cells,alteration in the pattern of cytokine secretion of these cells and the inhibition of differentiation of Th1 and Th17 cells.Thus these experimental results may provide the experimental basis for the treatment of EAE with triptolide and for the new drug development of multiple sclerosis.