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目的探讨丝裂原活化的蛋白激酶(MAPK)信号通路在B7H1-Ig诱导Ⅰ型调节性T细胞(Tr1)分化过程中的作用。方法以预包被而固相化的小鼠B7H1-Ig融合蛋白加抗CD3单克隆抗体(单抗)刺激新鲜分离的C57BL/6小鼠初始CD4+CD62L+T细胞,诱导其向Tr1细胞分化。Western blotting检测MAPK信号通路(ERK1/2、p38 MAPK、JNK)的活化状况。在B7H1-Ig开始刺激时分别加入ERK1/2、p38和JNK通路特异性抑制剂PD98059、SB203580和SP600125,分别采用ELISA法、混合淋巴细胞反应(MLR)、流式细胞术和Western blotting分析检测抑制MAPK信号对B7H1-Ig刺激的CD4+T细胞产生的细胞因子分泌格局、功能及Foxp3表达的影响。结果 B7H1-Ig联合抗CD3单抗激活并诱导一群IL-10+IFN-γ+IL-5+IL-4low/-IL-2low/-Foxp3-Tr1细胞,通过分泌抑制性细胞因子IL-10发挥免疫抑制功能。Western blotting检测结果显示B7H1-Ig可激活CD4+T细胞中的p38 MAPK信号通路,对ERK1/2和JNK信号通路无明显影响。抑制p38 MAPK活性,可使B7H1-Ig诱导的CD4+T细胞IL-10和IL-5的分泌减少、免疫抑制功能减弱,促进B7H1-Ig刺激的CD4+T细胞向CD25+Foxp3+调节性T细胞(Treg)分化。结论 p38 MAPK信号通路的活化或抑制是参与调控由B7H1-Ig诱导的Tr1细胞分化及其与CD4+CD25+Foxp3+Treg之间相互转化的重要分子机制。
Objective To investigate the role of mitogen-activated protein kinase (MAPK) signaling pathway in the differentiation of type I regulatory T cells (Tr1) induced by B7H1-Ig. Methods Freshly isolated C57BL / 6 mouse primary CD4 + CD62L + T cells were stimulated with precoated and immobilized mouse B7H1-Ig fusion protein plus anti-CD3 monoclonal antibody (mAb) to induce differentiation into Tr1 cells . The activation of MAPK signal pathway (ERK1 / 2, p38 MAPK, JNK) was detected by Western blotting. When B7H1-Ig started to be stimulated, ERK1 / 2, p38 and JNK pathway-specific inhibitors PD98059, SB203580 and SP600125 were respectively added and detected by ELISA, mixed lymphocyte reaction (MLR), flow cytometry and Western blotting analysis Effects of MAPK Signaling on Cytokines Production, Function and Foxp3 Expression Induced by B7H1-Ig in CD4 + T Cells. Results B7H1-Ig combined with anti-CD3 monoclonal antibody activated and induced a group of IL-10 + IFN-γ + IL-5 + IL-4low / -IL-2low / -Foxp3-Tr1 cells to secrete inhibitory cytokines IL- Immunosuppressive function. Western blotting results showed that B7H1-Ig could activate p38 MAPK signal pathway in CD4 + T cells, but had no effect on ERK1 / 2 and JNK signaling pathway. Inhibition of p38 MAPK activity decreased the secretion of IL-10 and IL-5 from CD4 + T cells induced by B7H1-Ig, decreased the immunosuppressive function and promoted the B7H1-Ig-stimulated CD4 + T cells to CD25 + Foxp3 + regulatory T cells (Treg) differentiation. Conclusion Activation or inhibition of p38 MAPK signaling pathway is an important molecular mechanism involved in the regulation of B7H1-Ig-induced Tr1 cell differentiation and its interaction with CD4 + CD25 + Foxp3 + Tregs.