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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 To study the effect of human cytomegalovirus (HCMV) infection on the cell cycle of ARPE-19 cells and to observe the relationship between HCMVIE2 and HCMVIE2 gene and explore the pathogenesis of HCMV infection. Methods Synchronized ARPE-19 was infected with HCMV as the infection group and the mock-infected group as control group. The cells were harvested at 1, 2, 3, 4 and 5 days after infection. The expression of HCMV IE mRNA was detected by RT-PCR. The expression of HCMV IE protein was detected by immunofluorescence. The expression of HCMV early protein IE And the expression of late protein pp65. The IE2 fragment was amplified by PCR from plasmid pIEP86AD169. The eukaryotic expression plasmid pEGFP-N3-IE2 was constructed and transfected into ARPE-19 cells by lipofectamine. The expression of IE2 gene was analyzed by RT-PCR and immunofluorescence. Flow Cytometry to detect changes in cell cycle. Results HCMV IE72, IE86 and pp65 could detect the expression of ARPE-19 cells at days 2, 3 and 5, respectively. Flow cytometry showed that the infected cells were infected with S phase cells (P <0.05) .PEGFP-N3-IE2 transiently transfected the cell cycle showed a significantly increased proportion of S phase cells.Conclusion ARPE-19 is allowed by HCMV Cells, HCMV can cause ARPE-19 cell cycle changes, this change and the expression of IE2 gene has a certain relationship.