鼠巨细胞病毒干扰神经干细胞分化相关基因表达的体外研究  

The experimental study on murine cytomegalovirus interferes the differentiation related genes expression of neural stem cells in vitro

作　　者：田佳[1] 刘兴楼[2] 方峰[2] 王慧[2] 张慧娟[2] 罗丹[2] 周玉峰[2] 李革[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,武汉430030 [2]深圳市妇幼保健院,518028

出　　处：《中华微生物学和免疫学杂志》2011年第5期390-397,共8页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30671859）

摘　　要：目的研究鼠巨细胞病毒（MCMV）对体外培养神经干细胞（NSCs）wnt信号通路下游分化相关靶基因表达的影响，以探讨CMV先天感染致胎脑发育异常的分子机制。方法体外分离培养BALB／c胎鼠NSCs，用感染复数（MOI）为5、1和0．1的MCMV Smith毒株感染NSCs并进行分化培养，Western blot法检测病毒感染后NSCs Wnt信号通路下游分化相关靶基因c-myc、cyclinD1、ngn-1、ngn-2蛋白表达水平的动态变化，real-timeRT-PCR检测病毒感染后NSCs Wnt信号通路关键分化基因ngn-1mRNA水平的动态变化。结果感染组c-mye蛋白表达量在分化培养后第0．5-5天均显著低于正常对照组（P〈0．05）；感染组cyclinD1蛋白表达量在第0．5天和1天显著低于正常对照组（P〈0．05）；在第2天和3天显著高于正常对照组（P〈0．05）；在第4天和5天，MOI=5组显著低于其他3组（P〈0．05）；感染组ngn-1蛋白表达量在第1～5天显著低于正常对照组（P〈0．05）；感染组ngn-1mRNA水平在第0．5～5天均显著低于正常对照组（P〈0．05）；感染组ngn-2蛋白表达先降后升，与正常对照组先升后降相反，MOI=0．1和1组峰值后移至感染后第5天，且明显低于正常对照组峰值（P〈0．05）。MOI=5组未表现出明显的峰值，各感染组ngn-2表达量在感染后1d明显低于正常对照组（P〈0．05）；感染后2d，MOI=5组明显低于正常对照组（P〈0．05）；在感染后3、4、5d，各组ngn-2表达量又明显高于正常对照组（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感染明显抑制NSCs Wnt信号通路分化相关基因c-myc和ngn-1蛋白表达，抑制ngn-1mRNA表达，并诱导cyclinD1和ngn-2蛋白表达紊乱，其效应随感染滴度增加而更为显著。MCMV抑制和干扰NSCs Wnt信号通路下游分化相关靶基因表达可能是其抑制NSCs增殖分化进而导致胎脑损伤的重要作用机制之一。Objective To investigate the influence of murine cytomegalovirus （MCMV） infection on the expression of downstream differentiation related target genes of Wnt signaling pathway in neural stem cells （NSCs） in vitro and explore the molecular mechanism of fetal encephalodysplasia caused by CMV infection. Methods NSCs were separated from fetal BALB/c mouse and cultured in vitro. The NSCs infected by MCMV at a MOI （multiplicity of infection） of 5, 1 and 0.1, respectively, were cultured in differentiation medium. The dynamic expression of the downstream differentiation related target genes （c-myc, cyclinD1, ngn-1 and ngn-2） of Wnt signal pathway in NSCs were measured by Western blot. Real-time RT-PCR was employed to measure the expression levels of the key differentiation genes ngn-1 in Wnt signal pathway of NSCs post infection. Results The protein levels of c-myc in the infected groups were significantly lower than that in the normal control at 0.5-5 d （P 〈0.05） ; At 0.5 d and 1 d post-infection （p. i. ）, the protein levels of cyclinD1 in the infected groups were lower than that in the normal control （P 〈 0.05）. At 2 d and 3 d p. i. , the cyclinD1 expression in the infected groups was higher than that in the control group （P 〈 0.05）. However, at 4 d and 5 d p. i. , the cyclinD1 levels in the group of the MOI of 5 were lower than inother three groups （P 〈 0.05 ）. The expression of ngn-1 protein in the infected groups was reduced importantly compared with normal control at 1-5 d p.i. （ P 〈 0.05 ）. The expression of ngn-1 mRNA in the infec- ted groups was lower than that in the control group at all time points （ P 〈 0.05 ）. The expression of ngn-2 protein decreased at first and then increased, which was opposite to the normal control. The peak of ngn-2 expression in groups of the MOI of 0.1 and 1 occurred later and were significantly lower than that in the normal control （ P 〈 0.05 ）. No distinct peak was seen in the group of the MOI of 5. At 1 d p. i. , the ex

关 键 词：巨细胞病毒 神经干细胞 WNT信号通路 C-myc CYCLIND1 Ngn.1 Ngn-2 

分 类 号：R3[医药卫生—基础医学]