调节性T细胞在小鼠巨细胞病毒感染胚胎成纤维细胞机制中的免疫调控作用

目的 体外研究调节性T细胞（Treg）在小鼠胚胎成纤维细胞（MEF）感染小鼠巨细胞病毒（MCMV）机制中的免疫调节作用。方法 采用尼龙毛柱和MACS Treg磁珠法获得纯化的Treg和去除Treg的T细胞（TdepTreg），建立TdepTreg和添加Treg的T细胞与感染MCMV的MEF（MEFMCMV）体外共培养细胞模型。用蚀斑法检测共培养细胞上清中感染性病毒量；流式细胞术检测共培养体系中Tc1(CD3+CD8+IFN-γ+)，Tc2（CD3+CD8+IL4+），Th1（CD3+CD8-IFN-γ+），Th2（CD3+CD8-IL-4+）细胞比率变化；双抗体夹心ELISA法检测共培养细胞上清中转化生长因子-β（TGF-β）和IL-10蛋白表达水平。结果 TdepTreg与MEFMCMV共培养3 d后上清中感染性病毒量较感染对照组显著降低，同时效应性T细胞各亚群比率较非感染对照组显著增加。向TdepTreg-MEFMCMV体系中添加Treg可增加共培养体系中感染性病毒量，同时Tc1、Tc2和Th1细胞比率显著减少，且均与Treg比率存在剂量相关性；而Th2比率仅在Treg比率增至20%时显著低于感染对照组。添加Treg的共培养体系中IL-10和TGF-β蛋白表达水平随Treg比率增加而增高，与Treg存在正性相关。结论 Treg可通过IL-10、TGF-β等途径抑制效应性T细胞的免疫保护作用，并且由于Treg抑制作用的不均衡性，导致Th1/Th2和Tc1/Tc2平衡失调，创造了一个利于CMV增殖的免疫环境。

Immune regulation of regulatory T cells in cytomegalovirus infection of mouse embryo fibroblasts

Objective To explore the role of regulatory T cells in murine cytomegalovirus (MCMV) infection of mouse embryonic fibroblasts (MEF). Methods A co culture system of T cells and MCMV infected MEF (MEFMCMV) was established. T cells were isolated from spleens of BALB/c mice infected by MCMV two months ago. CD3 T cells were purified by nylon wool column method and confirmed by flow cytometry. Treg and TdepTreg(T cells depleted of Treg) were isolated from T cells purified by MACS Treg Isolation Kit and confirmed by flow cytometry. To understand whether regulatory T cell (Treg) inhibits antiviral immune responses of T cells, Treg addition and Treg removal experiments were performed. In the co-culture system, the rates of effector T cell (Te) subpopulations, including Tc1 (CD3+CD8+IFN-γ+)，Tc2 (CD3+CD8+IL4+)，Th1 (CD3+CD8-IFN-γ+)，Th2 (CD3+CD8-IL-4+) were detected by flow cytometry; The levels of Treg cytokines, IL-10 and TGF-β of co culture supernatants were measured by double antibody sandwich ELISA; influence of co cultured T cells on MCMV replication was determined by measuring the viral load of MCMV in co culture supernatants using plaque assay. Results Treg removal study showed, viral load of TdepTreggroup was markedly lower than that of MCMV infected MEF group, and the proportions of Th1, Tc1 and Tc2 were increased significantly compared with those of infected control. In Treg addition study, adding Treg to co culture system in proportion could increase viral load; meanwhile, the percentages of Tc1, Tc2 and Th1 were obviously lower than that of TdepTreg-MEFMCMVgroup, and the percentage of Th2 was not significantly decreased until Treg percentage reached 20% (P<0.05); The percentage of Treg was inversely correlated with those of Th1, Tc1 and Tc2 cells (P<0.01); Moreover, the expressing levels of both IL 10 and TGF β in Treg adding groups were significantly increased compared with TdepTreg-MEFMCMV groups. Conclusions Treg appeared to down regulate the proportions of Tc1, Tc2, Th1 and Th2, and the disproportion inhibition of Treg led to the disturbance of Th1/Th2 and Tc1/Tc2 ratios, which benefited for CMV replication.