人巨细胞病毒及其IE86蛋白对ARPE-19细胞周期影响的研究

发布时间：2018-04-18 11:07

本文选题：人巨细胞病毒 + ARPE-19　；参考：《青岛大学》2010年硕士论文

【摘要】： 目的：研究人巨细胞病毒(human cytomegalovirus, HCMV)感染人视网膜色素上皮细胞ARPE-19后对其细胞周期的影响,研究这种影响与HCMV IE86基因的相关性,探讨HCMV感染导致巨细胞病毒性视网膜炎的发病机制。方法：用HCMV感染同步化于Go/G1期的ARPE-19细胞作为感染组,同时设未感染组为对照组。于感染后1d、2d、3d、4d和5d收获细胞,RT-PCR方法检测HCMVIE72、IE86 mRNA水平的表达,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IE72、IE86和晚期蛋白pp65的表达。MTT法检测病毒感染1-6天后对细胞增殖的影响,流式细胞术检测感染组与未感染组细胞DNA含量的变化。Western blot法检测病毒感染后细胞Cyclin D1、CyclinE表达的变化。采用PCR方法从质粒pIEP86AD169扩增出IE86基因片段,构建真核表达质粒pEGFP-N3-IE86,脂质体介导瞬时转染ARPE-19细胞,RT-PCR、免疫荧光法分析IE86基因的表达。流式细胞术检测质粒转染组及未转染组细胞DNA含量的变化。结果：HCMV IE72的mRNA在病毒感染后第1天检测到开始表达,IE86的mRNA在感染后第3天检测到开始表达；HCMV IE72、IE86和pp65蛋白可分别在病毒感染ARPE-19细胞2天、3天和5天时开始表达。MTT检测显示病毒感染3-6天后OD值分别为(0.646-0.0558)、(0.664±0.0765)、(0.723±0.1474)和(0.803±0.1073),与对照组比较,差异均有统计学意义(P0.05)。流式细胞术示感染组在感染后第3天、4天、5天细胞周期的S期细胞比率为(21.78%±2.65%),(26.65%±0.71%),(31.80%±3.97%),与对照组比较,差异均有统计学意义(P0.05)。Cyclin D1在未感染细胞及病毒感染1-2天时高表达,自感染3天起表达量开始下降。Cyclin E在未感染细胞及病毒感染1天时呈低表达状态,自感染第2天起表达量开始上升。瞬时转染质粒pEGFP-N3-IE86后24小时可检测到IE86在]mRNA水平和蛋白水平的表达。质粒瞬时转染48小时后细胞周期中S期细胞比率为(20.74%±1.60%),与对照组比较,差异有统计学意义(P0.05)。结论：HCMV可感染ARPE-19细胞。HCMV可促同步化于Go/G1期的ARPE-19细胞进入S期,且停滞于S期。IE86蛋白可以引起同步化于Go/G1期的ARPE-19细胞进入S期,提示HCMV导致ARPE-19细胞周期的改变可能由HCMV的IE86蛋白引起。
[Abstract]:Aim: to study the effect of human cytomegalovirus (HCMV) on the cell cycle of human retinal pigment epithelial cells (ARPE-19), and to investigate the relationship between the effect and HCMV IE86 gene, and to explore the pathogenesis of cytomegalovirus retinitis caused by HCMV infection.Methods: HCMV infected ARPE-19 cells in Go/G1 phase were used as infection group and non-infected group as control group.RT-PCR was used to detect the expression of HCMV IE72nIE86 mRNA in the harvested cells on the 1st day, 2nd day, 3rd day and 5th day after infection.The expression of HCMV IE protein in cell nucleus was detected by immunofluorescence assay. The expression of HCMV early protein IE72 IE86 and late protein pp65 was detected by Western blot method. The effect of virus infection on cell proliferation was detected by MTT assay after 1-6 days of infection.The changes of DNA content in infected and uninfected cells were detected by flow cytometry. The expression of Cyclin D1 cyclin E was detected by Western blot assay.An eukaryotic expression plasmid pEGFP-N3-IE86 was constructed by amplification of IE86 gene fragment from plasmid pIEP86AD169 by PCR method, and transient transfection of ARPE-19 cell line RT-PCR was carried out by liposome. The expression of IE86 gene was analyzed by immunofluorescence.The changes of DNA content in plasmid transfected group and untransfected group were detected by flow cytometry.Results on the first day after virus infection, the mRNA of mRNA of IE72 was detected and the mRNA that began to express iE86 was detected. On the third day after infection, the expression of HCMV IE72, IE86 and pp65 protein began to express on the 2nd day and 5th day after the virus was infected with ARPE-19 cells, respectively, and the expression of pp65 protein began on the 2nd day and 5th day after the virus infection.The OD values were 0.664 卤0.0765 卤0.1474 and 0.803 卤0.1073 respectively after 3-6 days of virus infection, which were compared with those of the control group.The difference was statistically significant (P 0.05).Flow cytometry showed that the S phase cell ratio of the infected group was 21.78% 卤2.65% 卤2.65% 卤0.71% 卤31.80% 卤3.97% on the 3rd day after infection, and the difference was statistically significant compared with the control group. The expression of Cyclin D1 was significantly higher than that of the control group at 1-2 days after infection.The expression of Cyclin E began to decrease after 3 days of infection. The expression of Cyclin E began to increase on the second day of infection.The expression of IE86 at mRNA and protein levels was detected 24 hours after transient transfection of plasmid pEGFP-N3-IE86.The ratio of S phase cells in the cell cycle was 20.74% 卤1.60% after transient transfection 48 hours after transfection, and the difference was statistically significant compared with the control group (P 0.05).ConclusionHCMV can infect ARPE-19 cells. HCMV can induce ARPE-19 cells synchronized in Go/G1 phase to enter S phase, and arrest in S phase. IE86 protein can induce ARPE-19 cells synchronized in Go/G1 phase to enter S phase.The results suggest that the change of ARPE-19 cell cycle induced by HCMV may be caused by the IE86 protein of HCMV.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

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