HCMV感染对HEL细胞MCM装载因子Cdc6与Cdt1的影响

发布时间：2018-01-13 11:46

  本文关键词：HCMV感染对HEL细胞MCM装载因子Cdc6与Cdt1的影响　出处：《中南大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 人巨细胞病毒 HEL细胞 Cdc6 Cdt1

【摘要】： 目的研究人类巨细胞病毒(human cytomegalovirus，HCMV)感染对人胚肺成纤维(human embryonic lung fibroblastic，HEL)细胞的MCM装载因子Cdc6与Cdt1表达的影响，从分子水平上探讨HCMV的发病机制。 方法用血清饥饿法同步化HEL细胞于G_0／G_1期，流式细胞术测细胞周期；用HCMV感染同步化的HEL细胞作为感染组，同时设模拟感染组为对照组。用倒置相差显微镜观察两组细胞形态改变至感染后7天。于感染后12h(hour)、24h、48h、72h和96h收获细胞，提取总的RNA，采用半定量逆转录—聚合酶链反应(RT—PCR)技术检测Cdc6 mRNA的表达水平；用免疫细胞化学法检测两组细胞核内HCMV Cdt1蛋白。 结果 1．模拟感染组未见细胞形态学改变，感染组感染后36h HEL细胞有形态学的改变即可见局部HEL细胞变圆、胞体变大、膨胀，7天时形态学的改变更加明显，所有细胞均发生病变。 2．12h、24h、48h、72h和96h两组HEL细胞Cdc6 mRNA表达水平均有显著性差异(P＜0.05)，12h、24h和48h时感染组较模拟感染组明显增加，72h和96h时模拟感染组较感染组明显升高。感染组在感染后12h时Cdc6 mRNA表达水平最高，后逐渐下降，至72h时达最低水平，96h时又稍有回升；模拟感染组在感染12h时Cdc6mRNA表达水平最高，后逐渐下降，至48h时达最低水平，72h时后开始回升，96h时继续回升。 3．12h、24h、和96h两组HEL细胞Cdt1蛋白水平均有显著性差异(P＜0.05)，而48h、72h两组HEL细胞Cdt1蛋白水平无明显差异(P＞0.05)。12h和96h时感染组较模拟感染组明显降低，24h感染组较模拟感染组明显升高。模拟感染组在感染后12h时Cdt1蛋白表达水平最高，后逐渐下降，至24h时达最低水平，96h又稍有回升；感染组在感染后12h Cdt1蛋白表达水平较低，24h达到最高峰，48h时急剧下降，一直维持较低的稳定水平。 结论 1．HCMV诱导Cdc6 mRNA过度表达，导致Cdc6的积聚，最终将影响细胞周期的进程。 2．HCMV诱导Cdt1蛋白表达延迟，从而影响了MCM复合物的装载和pre-RC的形成，使细胞停滞在G_1期，阻止DNA的复制。
[Abstract]:Objective to study human cytomegalovirus cytomegalovirus. Human embryonic lung fibroblastic was infected with human fetal lung fibroblasts. The effects of MCM loading factor Cdc6 and Cdt1 on the expression of HCMV in Hell cells were studied in order to explore the pathogenesis of HCMV at molecular level. Methods the HEL cells were synchronized by serum starvation in the G _ 0 / G _ S _ 1 phase and the cell cycle was measured by flow cytometry. The synchronous HEL cells infected with HCMV were used as the infection group. At the same time, the simulated infection group was set up as the control group. The morphologic changes of the cells in the two groups were observed by inverted phase contrast microscope until 7 days after infection, and 24 hours and 48 hours after infection. The cells were harvested at 72 h and 96 h, the total RNAs were extracted and the expression level of Cdc6 mRNA was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The HCMV Cdt1 protein in the nuclei of the two groups was detected by immunocytochemistry. Results 1. There were no morphological changes in the simulated infection group, but there were morphological changes in the HEL cells 36 hours after infection. The local HEL cells became round, the cell bodies became larger and expanded. At 7 days, the morphological changes were more obvious, and all the cells had pathological changes. There was a significant difference in the expression of Cdc6 mRNA between the two groups (P < 0.05) and the expression of Cdc6 mRNA at 24 h (P < 0.05) and 96 h (P < 0.05). At 24h and 48h, the number of infected group was significantly higher than that of simulated infection group. At 72 h and 96 h, the expression of Cdc6 mRNA in the simulated infection group was significantly higher than that in the infected group. The expression of Cdc6 mRNA in the infected group reached the highest level at 12 h after infection, then decreased gradually, and reached the lowest level at 72 h after infection. At 96 h, there was a slight rise. In the simulated infection group, the expression of Cdc6mRNA reached its highest level at 12h, then decreased gradually, and continued to rise again at 48h after reaching the lowest level at 72h and after 96h. The levels of Cdt1 protein in HEL cells at 3.12h and 96h were significantly different (P < 0.05, P < 0.05), but at 48h, the levels of Cdt1 protein in HEL cells were significantly higher than those in control group (P < 0.05). There was no significant difference in Cdt1 protein level in HEL cells between the two groups at 72 h after infection (P > 0.05. 12 h and 96 h). The Cdt1 protein level in infected group was significantly lower than that in simulated infection group. The expression of Cdt1 protein in the simulated infection group was the highest at 12 h after infection, then decreased gradually, and then increased slightly at the lowest level at 24 h after infection. In the infected group, the expression of Cdt1 protein decreased sharply at 12 h after infection and reached the peak at 24 h, and maintained a low and stable level at 48 h after infection. Conclusion 1. HCMV-induced overexpression of Cdc6 mRNA leads to the accumulation of Cdc6, which will eventually affect the process of cell cycle. 2. HCMV-induced delayed expression of Cdt1 protein, which affected the loading of MCM complex and the formation of pre-RC. HCMV-induced cell arrest in G _ (1) phase and blocked the replication of DNA.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373

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