摘要
目的研究人巨细胞病毒(human cytomegalovirus,HCMV)感染ARPE-19细胞后对其细胞周期的影响,观察这种变化与HCMVIE2基因的关系,探讨HCMV感染的发病机制。方法用HCMV感染同步化的ARPE-19作为感染组,同时设模拟感染组为对照组。于感染后第1、2、3、4、5天收获细胞,RT-PCR方法检测HCMV IE mRNA的表达水平,用免疫荧光法检测细胞核内HCMV IE蛋白的表达,Western blot法检测HCMV早期蛋白IE和晚期蛋白pp65的表达。采用PCR方法从质粒pIEP86AD169扩增出IE2片段,构建真核表达质粒pEGFP-N3-IE2,脂质体介导瞬时转染ARPE-19细胞,RT-PCR、免疫荧光法分析IE2基因的表达。流式细胞术检测细胞周期的变化。结果 HCMV IE72、IE86和pp65可分别在病毒感染ARPE-19细胞后第2、3和5天时检测到表达,流式细胞术示感染组在感染第3、4、5天细胞周期的S期细胞比例显著增高,与对照组比较差异均有显著性统计学意义,P(0.05。瞬时转染质粒pEGFP-N3-IE2后细胞周期表现为S期细胞比例显著增高。结论 ARPE-19是HCMV的允许细胞,HCMV可引起ARPE-19细胞周期的改变,这种变化与IE2基因的表达有一定的关系。
Objective The aim of this study was to investigate the influence of human cytomegalovirus（HCMV） infection on the cell cycles of ARPE-19,observe the relationship of its influence with HCMV IE2 genes,and explore the pathogenesis of HCMV infection.Methods Synchronized ARPE-19 cells were infected with HCMV,and a mock-infection group was set up as the control.The infected ARPE-19 cells were harvested at 1,2,3,4 and 5 d after the infection.HCMV IE proteins and pp65 were detected by RT-PCR,immunofluorescence assay and Western blot.The eukaryotic expression plasmid pEGFP-N3-IE2 was constructed and transfected into the ARPE-19 cells with Lipofectamine 2000.The expression of IE2 in the ARPE-19 cells was detected by RT-PCR and immunofluorescence assay,and the cell cycles of ARPE-19 was analyzed by flow cytometry.Results The HCMV IE72,IE86 and pp65 were expressed at 2,3 and 5 d respectively after the infection.The HCMV infection group showed a significant increase in the number of cells in the S phase at 3,4 and 5 d as compared with the mock-infection group（P0.05）.And there was a significantly increased proportion of the S-phase cells after the transient pEGFP-N3-IE2 transfection.Conclusion ARPE-19 cells are the permissive cells of HCMV,and HCMV infection influences the cell cycles of ARPE-19 and arrests the cell cycle in the S phase,which may be induced by the IE2 gene.
出处
《医学研究生学报》
CAS
2010年第5期452-455,共4页
Journal of Medical Postgraduates
基金
国家自然科学基金(30770105)
青岛市科技计划项目(08-1-3-30-jch)
