HCMV感染对HEL细胞周期与MCM2、MCM8表达的影响

发布时间：2018-09-04 17:21

【摘要】： 目的研究人类巨细胞病毒(human cytomegalovirus,HCMV)感染对HEL细胞周期、MCM2和MCM8表达的影响，探讨HCMV感染可能的发病机制。方法用HCMV感染血清饥饿法同步化的人胚肺成纤维细胞(human embryonic lung fibroblastic,HEL)。HCMV感染作为感染组，同时设非感染组为对照组，用流式细胞仪检测细胞周期。用倒置相差显微镜观察两组细胞形态学改变至感染后7天。于HCMV感染后12h(hour)、24h、48h、72h和96h收获细胞，提取总RNA，采用半定量逆转录—聚合酶链反应技术(RT—PCR)检测HEL细胞MCM2和MCM8的表达水平。结果 1、非感染组未发现细胞形态学改变。感染组感染HCMV 48h后，可见细胞变形显著，部分梭形细胞变成突起丰富的单个球形细胞，72h后病变更加明显，细胞绝大部分变成圆形、椭圆形，胞体及胞核增大，胞质混浊，细胞透光性降低。7天时可见所有感染细胞均发生类似病变。 2、感染组在感染后12h有71.4％的HEL细胞处于G_1期，24h仍有70.2％的细胞处于G_1期。非感染组在感染后12h有63.5％的HEL细胞处于G_1期，24h有45.5％的细胞处于G_1期。两组比较，均有显著性差异(P＜0.01)。 3、12h、24h、48h、72h、96h两组细胞MCM2和MCM8 mRNA表达水平均有显著性差异(P＜0.05)，12h和24h时感染组表达水平低于非感染组，48h、72h、96h时感染组表达水平明显高于非感染组。在上述5个时间点中，非感染组MCM2、MCM8 mRNA表达水平的高峰出现在感染后24h，而感染组表达水平的高峰则出现在感染后48h，较之有明显滞后。结论： 1、HCMV感染影响了HEL细胞周期，大部分HEL细胞停滞在G_1期。 2、HCMV感染能诱导MCM2及MCM8 mRNA表达异常，，使HEL细胞MCM2及MCM8 mRNA表达水平迟滞，这可能是HCMV感染导致细胞周期阻滞于G_1期的机制之一。
[Abstract]:Objective to study the effect of human cytomegalovirus (human cytomegalovirus,HCMV) infection on the expression of MCM2 and MCM8 in HEL cell cycle, and to explore the possible pathogenesis of HCMV infection. Methods Human embryonic lung fibroblasts (human embryonic lung fibroblastic,HEL). HCMV infected with HCMV were used as infection group and non-infected group as control group. Flow cytometry was used to detect cell cycle. The morphologic changes of the two groups were observed by inverted phase contrast microscope until 7 days after infection. The total RNA, was extracted from the cells harvested 12 h after HCMV infection at 24 h, 48 h and 96 h. The expression levels of MCM2 and MCM8 in HEL cells were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results 1. No morphological changes were found in the non-infected group. After infection with HCMV for 48 h, the cells deformed significantly, some fusiform cells became protruding single spherical cells and the lesion became more obvious 72 hours later, most of the cells became round, oval, the cell body and nucleus increased, and the cytoplasm was turbid. In the infected group, 71.4% of the HEL cells in the infected group were in GSCL phase 1 and 70.2% of the cells were still in GSCL phase 1 at 12 h after infection. In the non-infected group, 63.5% of the HEL cells were in phase 1 and 45.5% of the cells were in phase 1 at 12 h after infection. The two groups were compared, The expression levels of MCM2 and MCM8 mRNA in the two groups were significantly different (P < 0. 05). The expression level of MCM2 and MCM8 mRNA in the infected group was lower than that in the non infected group at 12 h and 24 h. The expression level of MCM2 and MCM8 mRNA in the infected group was significantly lower than that in the non infected group at 48 h and 72 h and 96 h after infection (P < 0. 05), and the expression level of MCM8 mRNA in the infected group was significantly lower than that in the non infected group at 12 h and 24 h (P < 0. 05). The level was significantly higher than that in non-infection group. In the above five time points, the peak of MCM2,MCM8 mRNA expression in the non-infected group was 24 hours after infection, while the peak in the infected group was 48 hours after infection, which was significantly delayed compared with that in the infected group. Conclusion: 1HCMV infection affects the cell cycle of HEL, and most of the HEL cells stagnate in G1 phase. 2HCMV infection can induce abnormal expression of MCM2 and MCM8 mRNA. The delayed expression of MCM2 and MCM8 mRNA in HEL cells may be one of the mechanisms of cell cycle arrest induced by HCMV infection.
【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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