鼠巨细胞病毒影响神经干细胞分化及分化基因的表达

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0.1 MCMV 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NSCs Wnt信号途径关键分化基因Neurog2、Myc及Ccnd1 mRNA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MCMV EA（早期抗原）的阳性表达；分化培养3-9 d，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1 d明显低于正常对照组（P<0.05)，感染组Myc mRNA表达水平在第1-4 d显著低于正常组（P<0.05)，感染组Ccnd1 mRNA水平在第0.5-1 d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NSCs Wnt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。

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

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 Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs at early stage of differentiation culture.RESULTS: NSCs isolated from embryonic mouse brains proliferated to form neurospheres, strongly expressed nestin and differentiated into NF-200 positive neurons or GFAP positive astrocytes. The infected NSCs did not adhere to the wall and appeared differentiation growths, but showed swollen gradually after differentiation culture. The nestin expression in the infected cells downregulated slowly and was higher than that in control groups(P<0.05). The GFAP and NSE expressions of the infected cells were lower than those in control groups(P<0.05). The early antigen(EA) of MCMV was always detected in the cells in infected groups. The ratios of nestin positive cells in infected groups were higher than those in control groups, but the ratios of GFAP and NSE positive cells of former were lower than that of the latter from 3rd to 9th d after differentiation culture(P<0.05). The levels of Neurog2 mRNA and Myc mRNA in infected groups were markedly lower than those in normal control groups on 1st d and from 1st to 4th d after differentiation culture, respectively(P<0.05). The levels of Ccnd1 mRNA of infected groups were obviously lower than those in normal control groups from 12th h to 1st d(P<0.05). These changes in infected groups became more obvious as MCMV MOI increased.CONCLUSION: MCMV significantly inhibits differentiation of NSCs to neurons and astrocytes, and leads to the decrease in differentiated cells. MCMV inhibits or interferes with the gene expression of Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs. The effect that MCMV inhibits the expressions of differentiation and the differentiation genes in NSCs shows dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering with the expression of differentiation gene in NSCs, which is possibly the one of primary causes of brain development disorders induced by congenital CMV infection.