Zhou Yu-feng, Fang Feng, Dong Yong-sui, Zhou Hua, Zhen Hong, Liu Jin, Li Ge
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Er Ke Za Zhi. 2006 Jul;44(7):505-8.
Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection.
NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR.
NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P < 0.01). At the same time, the rates of GFAP and NSE positive cells' in the infection group were significantly lower than those in the normal group (P < 0.01). The scanning densities of Wnt-1 was 0.14 +/- 0.03 in the infection group while 0.32 +/- 0.04 in the control group (t = 7.21, P < 0.01). The scanning densities of Ngn1 were 0.09 +/- 0.01 and 0.21 +/- 0.02 in the two groups (t = 10.7, P < 0.01).
These results suggest that MCMV infection could inhibit neuronal differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased expression of Wnt-1 and Ngn1 may be involved in the inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation, which may lead to a new strategy for preventing and treating brain abnormalities caused by CMV infection through regulating these two signal pathways.
巨细胞病毒（CMV）是宫内感染导致先天性中枢神经系统异常的主要感染原因。然而，这些脑部异常的确切发病机制尚未完全阐明。据报道，室管膜炎、脑室周围坏死和钙化是先天性CMV感染患儿脑部最常见的表现。由于已从脑室区鉴定出许多多能神经干细胞（NSC），该区域的NSC有可能是病毒感染的主要靶点，这似乎是导致脑部异常的主要原因。因此，本研究的目的是探讨鼠巨细胞病毒（MCMV）感染对神经干细胞体外分化的影响及其机制，以及在先天性巨细胞病毒感染所致脑部异常机制中的作用。
从胎鼠BALB/c制备神经干细胞，以感染复数（MOI）=1感染插入报告基因LacZ的重组MCMV RM461。感染后第2天，通过免疫组织化学和流式细胞术检测巢蛋白、胶质纤维酸性蛋白（GFAP）和神经元特异性烯醇化酶（NSE）阳性细胞的比例，观察MCMV感染对神经干细胞分化的影响。通过逆转录-聚合酶链反应（RT-PCR）检测MCMV感染对与神经分化相关的Wnt-1和神经生成素1（Ngn1）基因表达的影响。
从胚胎小鼠脑部分离的神经干细胞强烈表达巢蛋白，这是神经干细胞的特异性标志物，并且有能力分化为NF-200和NSE阳性神经元或GFAP阳性星形胶质细胞。在MOI=1时，流式细胞术检测结果显示，感染组巢蛋白阳性细胞比例[（62.2±1.8）%]高于正常组[（37.2±2.4）%]（t=4.62，P<0.01）。同时，感染组GFAP和NSE阳性细胞率显著低于正常组（P<0.01）。感染组Wnt-1的扫描密度为0.14±0.03，而对照组为0.32±0.04（t=7.21，P<0.01）。两组Ngn1的扫描密度分别为0.09±0.01和0.21±0.02（t=10.7，P<0.01）。
这些结果表明，MCMV感染可抑制神经元分化，这可能是先天性CMV感染时脑发育障碍的主要原因。Wnt-1和Ngn1表达降低可能参与了鼠巨细胞病毒感染对神经干细胞分化的抑制作用，这可能为通过调节这两条信号通路预防和治疗CMV感染所致脑部异常提供新策略。
