TLR4通过活化NF-κB途径对调节性T细胞的抑制功能起作用-免疫学专业论文.docxVIP

三峡大学硕士学位论文 三 峡 大 学 硕 士 学 位 论 文 PAGE IV万方数据 PAGE IV 万方数据 量较无刺激对照组降低，而胞核中则相应增加；anti-TLR4组则无明显改变。 结论：在不同微环境中，CD4+CD25+Tregs表面的膜型TLR4具有不同的作用，继 而对CD4+CD25+Tregs的抑制功能进行调节。当经TLR4的内源性配体HMGB1活化时， CD4+CD25+Tregs以分泌促炎性细胞因子IFN-γ为主，其抑制功能减弱；当与外源性配 体LPS相互作用时，IL-1β和IL-10的含量同时增多，其抑制功能增强；二者均与NF-κB 信号分子的活化相关。综上诉述，TLR4通过活化NF-κB途径对调节性T细胞的抑制功 能起作用。 关键词：调节性T细胞 Toll样受体4 NF-κB 脂多糖 高迁移率组蛋白1 Abstract Objective Native Regulatory T cells,CD4+CD25+Tregs,have a significant immune suppressive function.The activation of different types of TLRs makes different effects.In this study,we prepared to find out the changes of cytokines secreted after the membrane molecule TLR4 binding to different ligands,and its effects on the function of CD4+CD25+ Tregs.Moreover,exploring its possible mechanisms. Methods (1)CD4+CD25+Tregs were isolated from healthy human peripheral blood and detected the sorting purity. (2)Collecting the CD4+CD25+Tregs and setting them into two main groups,normal CD4+CD25+Tregs and TLR4 inhibited CD4+CD25+Tregs. Thereafter,detecting the RNA expression of IL-1β,IL-10,IFN-γ and TGF-β with Real-time quantitative PCR after CD4+CD25+Tregs were stimulated with different concentrations of LPS and HMGB1.What’s more,detecting the secretion of the cytokines above in the culture supernatant with ELISA.(3)Analysing the proliferation of CD4+T cells after they mixed culturing with the above processing CD4+CD25+Tregs.(4)Using Western-blotting to detect the expression of NF-κB after different groups of CD4+CD25+Tregs were stimulated by LPS and HMGB1. Results (1)The purity of positively sorted CD4+CD25+Tregs is no less than 82%. (2)In Non anti-TLR4 group,the RNA level and protein content of IL-1β, IL-10 and TGF-β of HMGB1 treated CD4+CD25+Tregs decrease significantly compared with the control(P ﹤0.05),while in anti-TLR4 group, they are higher than that of the corresponding control (P﹤0.05).The change trends of RNA level and protein content of IFN-γ are the opposite, they are significantly higher than