人巨细胞病毒感染对宿主细胞周期蛋白表达影响的实验研究

目的观察人巨细胞病毒（HCMV）感染对宿主细胞周期蛋白（Cyclins）表达的影响。方法用HCMV感染同步化于G0/G1期的人胚肺成纤维细胞（HEL）,分别于感染后0 h、3 h、6 h、12 h、24 h、48 h、72 h、96 h终止培养。用免疫蛋白印迹法（Western Blot）检测CyclinE、CyclinA、CyclinD1蛋白的表达。结果HCMV感染接触抑制细胞12 h时CyclinE蛋白被诱导,感染后24 h出现CyclinE峰值;HCMV不能诱导CyclinA蛋白表达;CyclinD1没有被诱导,且在感染后24 h开始下降。结论HCMV感染同步化于G0/G1期的细胞后,诱导CyclinE蛋白明显升高,激活CyclinE/Cdk2激酶,使细胞周期越过G1/S限制点,将细胞周期阻止于晚G1期。

Experimental study of the effect of human cytomegalovirus infection on the expression of cyclins

Objective:To observe the effect of human cytomegalovirus infection on expression of cyclines.Metheds:HCMV infected cell was established in vitro by incubating passage cultured HEL and HCMV AD169 strain with different titres.The cells were synchronized in the G0/G1 stage by contact inhibition and infected with strain AD169 of HCMV at an MOI of 5 PFU per cell.We harvested infected cells at different time 0h,3h,6h,2h,24h,48h,72h and 96h post infection.Then expression of proteins of cyclineE,cyclineA and cyclineD1 were determined with Western Blot.Results:CyclinE protein was induced 12h PI and peak induction occurred 24h PI in contact-inhibited cells.CyclinA protein expression was not induced in HCMV infected density-arrested cells.CyclinD1 was not induced by the virus.Rather,the abundanc of cyclinD1 decreased 24 h PI.Conclusion:The expression of cyclinE and activity of cyclinE/Cdk2 kinase are increased obviously in G0/G1 stage cells infected with HCMV,which may induce the cell cycle to overpass G1/S restriction point and make the cell cycle arrested in later G1 stage.