小鼠巨细胞病毒感染鼠胚胎成纤维细胞对共培养调节性T细胞增殖和活化的影响

Effects of murine cytomegalovirus infected mouse embryonic fibroblasts on the proliferation and activation of co-cultured regulatory T cells

目的研究小鼠巨细胞病毒（MCMV）感染对共培养体系中调节性T细胞（Treg）的诱导作用和Treg在抗病毒免疫中的调节作用。方法建立小鼠T淋巴细胞与感染CMV的同系小鼠胚胎成纤维细胞（MEF）体外共培养的细胞模型。蚀斑法检测共培养上清液中MCMV负荷量；细胞计数法分析体系中T淋巴细胞的扩增；流式细胞术检测共培养体系中CD4^＋CD25^＋Treg细胞比例，Western印迹法检测T淋巴细胞中Foxp3蛋白表达强度；RT—PCR法检测MEF中转化生长因子（TGF）-βmRNA表达水平；双抗体夹心ELISA法检测细胞培养上清液中TGF—β蛋白表达水平。组间差异采用单因素方差分析。结果T淋巴细胞与MCMV感染的MEF共培养1d和3d后，培养上清液中病毒负荷量较对照组显著减少；但是共培养6d后，T淋巴细胞的免疫保护作用消失，病毒量为（5．58±0．67）×10^3PFU/mL，与感染对照组的（6．05±0．34）×10^5PFU／mL比较，差异无统计学意义。CMV感染3d后，T淋巴细胞数由感染前的（2．02±0．05）×10^6／mL增至（2．25±0．13）×10^6／mL（P〈0．05）；但继续培养至6d时，T淋巴细胞数为（2．08±0．14）×10^6／mL，与3d时比较，差异无统计学意义。CD4^-CD25^-Foxp3^＋Treg细胞比例和Foxp3蛋白表达变化相似，均随MCMV感染时间延长而增加，在感染后6d分别增至对照组的2倍和3倍；TGF-β在MEF中的基因转录水平（A值）由感染前的1．09±0．13增至感染3d后的3．15±0．54，共培养上清液中的蛋白表达强度在感染后3d增至（3．85±0．32）μg／L，与感染前的（1．74±0．14）μg／L比较，差异有统计学意义（P〈0．05）。结论随感染时间延长，MCMV可能通过促进TGF-β表达等多种途径诱导Treg增殖活化。

Objective To explore the effects of murine cytomegalovirus （MCMV） infected mouse embryonic fibroblasts （MEF） on the proliferation and activation of regulatory T cell in vitro. Methods A co-culture system of T cell and MCMV infected MEF （T-MEF MCMV） was established. The viral load of supernatant was determined by plaque assay. The proliferation of T cells was observed with cell counting. The proportion of CD4^＋ CD25^- cells was measured by flow cytometry. The levels of Foxp3 protein were measured by Western blot. Reverse transcription polymerase chain reaction （RT-PCR） assay was utilized to determine whether MCMV infection of MEF influenced the level of transforming growth factor （TGF）-β mRNA. The level of TGF-β protein in supernatant was measured by double-antibody sandwich enzyme linked immunosorbent assay （ELISA）. The difference between T-MEF MCMV group and control group was assessed by one-way ANOVA. Results When T cells were co cultured with MCMV infected MEF for 1 day and 3 days, the viral load in supernatant decreased. But when co-cuhure lasted for 6 days, the antiviral effect obviously diminished, as the viral load [（5.58±0.67） × 10^5 PFU/mL] of the experimental group showed no statistic difference with MEF MCMV control group [（6. 05±0.34）× 10^5 PFU/mL]. When co-cultured with MCMV infected MEF for 3 days, T cell increased from pre culture level of [（2. 02 ± 0.05）× 10^6/mL] to （2.25 ± 0.13） × 10^5/mL （P〈0.05）. But when co culture lasted for 6 days, the number of T cell returned to （2.08±0.14） ×10^6/mL, which had no statistic difference with that of co culture for 3 days system. Both the expressions of Foxp3 protein and the proportion of CD4^ - CD25^＋ Foxp3^＋ Treg cells in T-MEF MCMV group were up-regulated as the infection time extended, which were two times and three times of the control group level, respectively. The mRNA level （A value） of TGF-β in MCMV infected MEF increased from baseline of 1.09 ± 0.13 to ,3.15 ± 0.54 on day 3 after infection. The expression of TGF-β in supernatant 3 days after infection was （3.85±0.32） μg/L, which was significantly higher than that before infection [（ 1.74 ± 0.14） μg/L, P 〈 0. 05]. Conclusions Activated T cells have antiviral effect. However, the function of T cells is rapidly inhibited after activation, which may be due to the expression of Foxp3 mRNA induced by MCMV infected MEF and increased CD4^＋ CD25^＋ Treg proportion of the co-cultured T cells. TGF-β level is significantly increased after CMV infection, which may be an important mechanism of Treg proliferation. MCMV may manipulate Treg to evade specific immune elimination and, as a result, to cause CMV replication.