小鼠巨细胞病毒感染对调节性T细胞扩增的影响及机制  

The effects and mechanisms of murine cytomegalovirus infection on regulatory T cell expansion

作　　者：李亚男[1] 周玉峰[2] 舒赛男[2] 刘兴楼[2] 葛海霞[2] 方峰[2] 

机构地区：[1]吉林大学第一医院儿科,吉林长春130021 [2]华中科技大学同济医学院附属同济医院儿科,湖北武汉430030

出　　处：《临床儿科杂志》2009年第9期860-865,共6页Journal of Clinical Pediatrics

基　　金：国家自然科学基金资助项目(No.30572345)

摘　　要：目的在体内外水平研究小鼠巨细胞病毒(MCMV)感染对调节性T细胞(Treg)扩增的影响及其可能的机制。方法建立全身播散型MCMV慢性感染模型,分别采用Western blot法和流式细胞术动态检测小鼠脾细胞中Foxp3的蛋白表达强度和CD4+CD25+Foxp3+Treg细胞比率;模拟MCMV感染细胞的免疫环境,建立T淋巴细胞与MCMV感染的小鼠胚胎成纤维细胞(MEF)共培养细胞模型,在培养前和共培养3d后检测T淋巴细胞中的Foxp3蛋白表达水平及CD4+CD25+Foxp3+Treg细胞比率,RT-PCR方法检测MEF中转化生长因子(TGF)-βmRNA表达水平,双抗体夹心ELISA方法检测共培养上清中TGF-β蛋白表达水平。结果整体研究发现,MCMV在感染慢性期可显著增加T淋巴细胞中Foxp3的蛋白表达水平和CD4+CD25+Foxp3+Treg细胞比率。体外细胞模型发现,MCMV感染在体外同样可以上调T细胞中的Foxp3蛋白表达和Treg细胞比率。TGF-β在MEF中的基因表达和上清中的蛋白表达在MCMV感染后显著增加。结论MCMV感染在体内外均可诱导Treg在数量上的扩增和功能上的活化;MCMV感染导致宿主细胞TGF-β表达水平升高,可能是其在外周诱导Treg种群扩增的重要机制之一。Objective To explore the effects and mechanisms of murine cytomegalovirus（MCMV） on regulatory T cells（ Treg） expansion in vivo and in vitro. Methods A chronic disseminated MCMV-infected mice model was established. The expression levels of Foxp3 and the proportions of CD4＋ CD25＋ Foxp3＋ Treg in splenocytes were measured by Western blot and flow cytometry,respectively. T cells were isolated from spleens of BALB /c mice,which were infected by MCMV two months before. Then CD3＋T cells were isolated and purified by nylon fiber column. The co-culture system of T cells and MCMV-infected mouse embryonic fibroblasts（ MEFs） was established to mimic immune environment of MCMV-infected cells in vitro. The influence of MCMV on Foxp3 protein expression;CD4＋ CD25＋ Foxp3＋ Treg ratios;the levels of TGF-β mRNA in MEF,and TGF-β production in supernatants were determined by Western blot,flow cytometry,RT-PCR assay and double-antibody sandwich ELISA,respectively,3 days before and 3 days post infection. Results In vivo,Foxp3 expression increased significantly with the extension of infection period. Similar changes of CD4 ＋ PCD5 ＋ Foxp3 ＋ Treg＇s proportion were found in mice splenocytes. In vitro,the Treg ratio and Foxp3 protein level were upregulated with MCMV infection,consistent with that of MCMV in vivo study. Levels of TGF-β protein in supernatants and TGF-β mRNA in MCMV-infected MEFs were significantly increased post infection. Conclusions MCMV infection could induce Treg proliferation and activation both in vivo and in vitro,possibly through up-regulating TGF-β expression.

关 键 词：巨细胞病毒 调节性T细胞 转化生长因子Β Foxp3蛋白 

分 类 号：R392[医药卫生—免疫学]