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先天感染致神经损伤的机制。方法体外分离培养和鉴定BALB/c胎鼠NSCs并检测其分化潜能,用感染复数(MOI)为5、1和0.1的MCMVSmith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、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)表达的变化,采用MCMV早期抗原(EA)示踪感染过程(MOI=1),real—time RT-PCR检测分化早期NSCsWnt信号途径关键分化基因wnt-3和Wnt-7amRNA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,并可进一步诱导分化为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,而GFAP和NSE表达显著低于正常对照组(P〈0.05),可检测到MCMVEA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Wnt-3 mRNA水平在分化培养后第1~2天显著低于正常对照组(P〈0.05),感染组Wnt-7a mRNA水平在第0.5~2天明显低于正常对照组(P〈0.05);感染组和正常对照组的差异随病毒MOI的增加而更加明显。结论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;下调或干扰NSCs Wnt信号途径分化基因Wnt4和Wnt-7a的表达;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;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先天感染致脑发育异常的重要机制之一。
Objective To investigate the influence of murine cytomegalovirus (MCMV) infection on differentiation and differentiation gene expression of neural stem cells (NSCs) in vitro 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 multiplicity of infection (MOI) equaled to 5, 1 and 0.1, respectively, were cultured in differentiation medium. The morphological changes of the cells were observed by inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expression changes 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 differentiation genes Wnt-3 and Wnt-7a in Wnt signal pathway of NSCs at early phage of differentiation culture. Results NSCs isolated from embryonic mouse brains could proliferate to form neurospheres and strongly express Nestin and differentiate into NF-200 positive neurons or GFAP positive astrocytes. The NSCs of the infected groups couldn't adhere to the wall and appear differentiation growth, but showed swollen gradually after differentiation culture. The nestin expression of the infected groups downregulated slowly and was higher than that of the control groups ( P 〈 0.05 ). The GFAP and NSE expression of the infected groups were lower than that of the control groups (P 〈 0.05). The EA of MCMV could be always detected in the cells of the infected groups. The ratios of nestin positive cells of the infected groups were higher than that of the control groups, but the ratios of GFAP and NSE positive cells of the former were lower than that of the latter from 3rd to 9th day after differentiation culture (P 〈 0.05 ). The levels of Wnt-3 mRNA and Wnt-7a mRNA of the infected groups were markedly lower than that of the control groups from 1st to 2rid day and from 12th hour to 2nd day after differentiation culture respectively (P 〈 0.05 ) . These changes of the infected groups became more obvious as MCMV MOI increased. Conclusion MCMV could inhibit significantly NSCs differentiate to neurons and astrocytes and lead to the decrease of differentiated cells. MCMV could inhibit or interfere with the gene expression of Wnt-3 and Wnt-7a in Wnt signal pathway of NSCs. The effect that MCMV inhibited the differentiation and the differentiation gene expression of NSCs showed dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering the differentiation gene expression of NSCs, which is possibly the one of primary causes of brain development disorders caused by congenital CMV infection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2009年第6期548-554,共7页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目(30671859)