摘要目的 通过细胞水平探讨CD4+CD25+调节性T细胞(Treg)在T淋巴细胞与感染小鼠巨细胞病毒(MCMV)的小鼠胚胎成纤维细胞(MEF)共培养体系中发挥的免疫作用.方法 建立小鼠T细胞与感染MCMV的同系MEF(MEFMCMV)体外共培养细胞模型.通过噬斑法检测共培养上清中感染性病毒量;Western blot方法检测辅助性T细胞亚群TH1/TH2特异性转录因子T-bet/GATA-3蛋白表达水平;ELISA法检测共培养上清中细胞因子IL-4、IL-10和IFN-γ表达水平.结果 去除Treg的T细胞(TdepTreg)与MEFMCMV共培养3 d后可显著减少上清中的感染性病毒量;同时THl/TH2上游特异性转录因子T-bet/GATA.3和下游细胞因子IL-4、IL-10和IFN-γ的蛋白质表达水平显著升高.向共培养体系中添加Treg后病毒负荷量显著增加;T-bet/GATA-3和IFN-γ蛋白表达水平下降;IL-10蛋白表达水平在Treg比率为1%～2%时与TdepTreg组比较无显著差异,在Treg比率增至5%～20%时较TdepTreg组显著增加;而IL-4表达水平与TdepTreg组比较无显著差异,上述效应均与Treg添加比率成剂量相关性.结论 巨细胞病毒感染小鼠成纤维细胞后能激活效应性T细胞增殖活化,而Treg可抑制效应性T细胞在MCMV感染中的免疫保护作用.

abstractsObjective To explore the role of CD4+CD25+regulatory T cells(Treg)in the co-culture svstem consisting of T cells and mouse embryo fibroblasts(MEF)infected with murine cytomegalovirus (MCMV)in vitro.Methods A co-culture system of T cells and MCMV infected MEF(MEFMCMV)was established.The viral load in the supernatants was determined by plaque assay.TH1/TH2 differentiation-specific transcription factors T-bet/GATA-3 were assayed by Western blot.The levels of cytokines IL-4,IL-10and IFN-γ in the co-culture supenatants were measured by double-antibody sandwich ELISA.Results After 3 d incubation,the viral load in the supernatants of TdepTreg-MEFMCMC group,in which the T cells depleted Treg(TdepTreg)were co-cultured together with MEFMVMV,was significantly lower than that in MEFMCMV group.And in the co-cultivation of MEFMCMV and T cells without Treg the expressions of T-bet/GATA-3,IL-4,IL-10 and IFN-γ incteased significantly.Compared with the virus content in the TdepTreg-MEFMCMV group,the MCMV load in the"add-on Treg group"increased and the levels of T-bet/GATA-3 and IFN-γ were lower,and the expression of IL-4 didn't show any significant difference. Compared with the level of IL-10 in the TdepTreg-MEFMCMV group,the IL-10 level in the"add-on TREG group"didn't show any significant differece when the Treg ratio among total T cells was 1%～2%,and increased significantly when the Treg ratio was more than 5%.These eflfects were all correlated with Treg ratios in a dose-dependent manner.Conclusion MCMV infected MEF can induce the proliferation and activation of effector T cells,but Treg can inhibit the T cell-mediated protective effect on MCMV infection.