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目的:观察鼠巨细胞病毒(MCMV)感染对体外培养神经干细胞(NSCs)细胞周期进程和cyclins表达的影响,探讨MCMV感染致脑发育异常的机制。方法:本实验体外分离、培养和鉴定BABL/C胎鼠NSCs,以感染复数(MOI)为5,1和0.1的MCMVsmith毒株感染NSCs并于感染后1,2,3,4,5,6d收集细胞,采用Cyclin/DNA双参数流式细胞术检测感染细胞cyclinA,cyclinB1,cyclinD1,cyclinE的表达和细胞周期时相的动态变化,观察MC-MV对感染NSCs细胞周期进程的影响。结果:体外分离培养的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GFAP阳性的星形胶质细胞和NF-200阳性的神经元;各感染组cyclinA,cyclinB1,cy-clinD1和cyclinE的表达均上调,其中MOI=0.1表达逐渐上升,在第6d达峰值,MOI=1组表达高峰在第4d,MOI=5组表达高峰在第3d;感染组G0/G1期细胞比率减少,S期和G2/M期细胞比率增加,其变化趋势与cyclins的表达基本一致,并随MOI的增加变化越明显。结论:Cyclin/DNA多参数流式细胞术可用于NSCs细胞周期的分析;MCMV可通过上调cyclinA,cyclinB1,cyclinD1,cyclinE的表达来影响NSCs细胞周期进程,并与MOI存在一定量效依赖关系;MCMV感染可诱导NSCs从G0/G1期进入S期,出现S期和G2/M期偏移和阻滞,影响NSCs细胞周期进程,这可能是CMV抑制NSCs增殖并导致先天性脑发育异常的重要机制之一。
Objective: To observe the effect of murine cytomegalovirus (MCMV) infection on cell cycle progression and cyclins expression in cultured neural stem cells (NSCs) in vitro and to explore the mechanism of MCMV-induced brain dysplasia. Methods: NSCs were isolated, cultured and identified from fetal BABL / C embryos in vitro and infected with MCMVsmith strains with MOIs of 5, 1 and 0.1. The NSCs were infected at 1, 2, 3, 4, The cells were collected and the expression of cyclinA, cyclinB1, cyclinD1 and cyclinE in the infected cells were detected by Cyclin / DNA double-parameter flow cytometry to observe the effect of MC-MV on the cell cycle progression of infected NSCs. Results: The NSCs cultured in vitro showed a globular growth pattern, the NSCs specific marker Nestin expression was positive and could be further differentiated into GFAP positive astrocytes and NF-200 positive neurons. The expressions of cyclin A, cyclinB1, The expression of cy-clinD1 and cyclinE were all up-regulated, the expression of MOI = 0.1 was gradually increased, reached its peak on the 6th day, the expression peak of MOI = 1 was on the 4th day and the peak of MOI = The percentage of cells decreased, while the percentage of cells in S phase and G2 / M phase increased. The change tendency was consistent with the expression of cyclins, and the change was more obvious with the increase of MOI. CONCLUSION: Cyclin / DNA multiparameter flow cytometry can be used to analyze the cell cycle of NSCs. MCMV can affect the cell cycle progression of NSCs by up-regulating the expression of cyclin A, cyclinB1, cyclinD1 and cyclinE and has a dose-dependent relationship with MOI. MCMV Infection can induce the NSCs to enter the S phase from the G0 / G1 phase, the S phase and the G2 / M phase shift and block, affecting the cell cycle progression of NSCs, which may be an important mechanism of CMV inhibiting NSCs proliferation and causing congenital brain development abnormalities one.