鼠巨细胞病毒影响神经干细胞分化及分化基因的表达  

Influence of murine cytomegalovirus infection on the differentiation and the differentiation genes expression in neural stem cells

作　　者：罗丹[1,2] 舒赛男[1] 周玉峰[1] 刘兴楼[1] 田佳[1] 王惠[1] 周华[1] 李革[1] 方峰[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,湖北武汉430030 [2]广州儿童医院感染科,广东广州510120

出　　处：《中国病理生理杂志》2009年第11期2173-2181,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30671859)

摘　　要：目的:研究鼠巨细胞病毒(MCMV)感染对体外培养神经干细胞(NSCs)分化及分化基因表达的影响,探讨CMV先天感染致脑发育异常的机制。方法:体外分离培养和鉴定BALB/c胎鼠NSCs,检测细胞分化潜能,用感染复数(MOI)为5、1和0.1MCMV smith毒株感染NSCs并进行分化培养,倒置显微镜下观察细胞形态学改变,流式细胞术检测分化细胞比率,免疫荧光法观察NSCs及其分化细胞标记物nestin、GFAP和NSE表达的变化,采用MCMV早期抗原(EA)示踪感染过程(MOI=1),实时定量RT-PCR检测分化早期NSCsWnt信号途径关键分化基因Neurog2、Myc及Ccnd1mRNA水平的动态变化。结果:体外培养的NSCs呈球样生长,神经干细胞特异性标记nestin表达阳性,并可进一步分化为NF-200阳性的神经元和GFAP阳性的星形胶质细胞;分化培养后,感染组NSCs不能贴壁分化生长并逐渐出现肿胀,细胞nestin表达下调缓慢并显著高于正常对照组,GFAP和NSE表达显著低于正常对照组(P<0.05),可检测到MCMVEA(早期抗原)的阳性表达;分化培养3-9d,感染组nestin阳性细胞比率显著高于正常对照组,GFAP和NSE阳性细胞比率显著低于正常对照组(P<0.05);感染组Neurog2 mRNA水平在分化培养后第1d明显低于正常对照组(P<0.05),感染组Myc mRNA表达水平在第1-4d显著低于正常组(P<0.05),感染组Ccnd1 mRNA水平在第0.5-1d明显低于正常组;感染组和正常组的差异随病毒MOI的增加而更明显。结论:(1)MCMV感染可明显抑制NSCs向神经元和星形胶质细胞方向分化,导致分化细胞比率减少;(2)MCMV可下调或干扰NSCsWnt信号途径分化基因Neurog2、Myc和Ccnd1的表达;(3)MCMV抑制NSCs分化及其分化基因表达的效应与MOI大小存在一定量效依赖关系;(4)MCMV可能通过抑制NSCs分化基因的表达来抑制其分化,这可能是CMV感染致脑发育异常的重要机制之一。AIM： The influence of MCMV infection on differentiation and differentiation gene expression in neural stem cells （NSCs） in vitro were investigated for studying the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. METHODS： NSCs were separated from fetal BALB/C mouse, and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunofluorescence. The NSCs infected by MCMV at dosage of MOI（ multiplicity of infection） equaled to 5, 1 and 0.1 ,respectively, were cultured in differentiation medium. The morphological changes of infected cells were observed under inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expressions of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence（ MOI = 1 ）. The expression of early antigen（EA） of MCMV was detected to observe the infection process. Real - time RT - PCR method was employed to measure the expression levels of the key genes Neu

关 键 词：巨细胞病毒 神经干细胞 细胞分化 基因 Neurog2 基因 MYC 基因 CCND1 

分 类 号：R373[医药卫生—病原生物学]