人巨细胞病毒感染对人胚肺成纤维细胞周期及复制前复合物的影响

发布时间：2018-01-02 05:23

  本文关键词：人巨细胞病毒感染对人胚肺成纤维细胞周期及复制前复合物的影响　出处：《中南大学》2008年博士论文　论文类型：学位论文

  更多相关文章： 人巨细胞病毒 人胚肺成纤维细胞 人巨细胞病毒IE_172蛋白 细胞周期 复制前复合物 Cdc10依赖性转录因子1 细胞分裂周期基因产物6 微小染色质维持蛋白2 微小染色质维持蛋白8 复制抑制因子Geminin

【摘要】： 目的 研究人巨细胞病毒(human cytomegalovirus,HCMV)感染人胚肺成纤维细胞(human embryonic lung fibroblast,HELF)后对其细胞周期及复制前复合物(pre- replication complex,pre-RC)的影响。探讨HCMV感染的发病机制。 方法 1、血清饥饿法同步化HELF于G_0/G_1期,用HCMV感染同步化的HELF作为感染组(细胞数=1×10~6),同时设模拟感染组为对照组(细胞数=1×10~6)。用倒置相差显微镜观察两组细胞形态学变化至感染后7天,于感染后12、24、48、72和96小时收获细胞。 2、流式细胞仪检测HELF细胞周期。 3、免疫细胞化学法检测两组HELF细胞核内HCMV IE_172蛋白,Cdt1蛋白水平。 4、提取总RNA,用半定量逆转录—聚合酶链反应(RT-PCR)检测两组HELF复制前复合物Cdt1 mRNA、Cdc6 mRNA、Mcm2 mRNA、Mcm8 mRNA的表达水平。 5、RT-PCR法检测两组HELF复制抑制因子Geminin的表达水平。 结果 1、两组HELF细胞形态学变化: 感染组HELF感染后72h时即可见局部细胞变圆,膨胀,细胞体及细胞核巨大化,7天时可见所有细胞均发生病变;模拟感染组HELF各时间点均未见细胞形态学改变。 2、两组HELF HCMV IE_172蛋白阳性产物表达: 感染组HELF于感染后12h时细胞核内即出现呈棕黄色颗粒的HCMV IE_172蛋白产物的表达;模拟感染组HELF各时间点均未出现HCMV IE_172蛋白阳性产物。 3、两组HELF细胞周期检测结果: 感染组在感染后12h时有70.4%的细胞处于G_1期,24h时有69.9%的细胞处于G_1期;模拟感染组在感染后12h时有65.7%的细胞处于G_1期,24h时有43.8%的细胞处于G_1期。感染组HELF停滞G_1期于较模拟感染组明显增多,两组比较,差异均有统计学意义(P＜0.01)。 4、两组HELF Cdt1 mRNA表达水平: 模拟感染组在感染后12h时Cdt1 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。感染组在感染后12h时Cdt1 mRNA表达水平较低,24h时稍有升高,48h达最高峰,72h时开始下降,96h时大致维持72h水平。两组比较,12h、24h和48h时Cdt1 mRNA表达水平差异均有统计学意义(P＜0.05),12h和24h时感染组较模拟感染组明显降低,48h时感染组较模拟感染组明显升高。模拟感染组Cdt1 mRNA表达水平的高峰出现在感染后12h,而感染组的高峰则出现在感染后48h,较模拟感染组明显滞后。 5、两组HELF Cdt1蛋白水平: 12h、24h、和96h时两组HELF Cdt1蛋白水平差异均有统计学意义(P＜0.05),48h、72h时两组HELF Cdt1蛋白水平差异无统计学意义(P＞0.05)。12h和96h时感染组较模拟感染组明显降低,24h时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时又稍有回升;感染组在感染后12h时Cdt1蛋白表达水平较低,24h时达到最高峰,48h时急剧下降,一直维持较低的稳定水平。 6、两组HELF Cdc6 mRNA表达水平: 12h、24h、48h、72h和96h时两组HELF Cdc6 mRNA表达水平差异均有统计学意义(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Cdc6 mRNA表达水平最高,后逐渐下降,至48h时达最低水平,72h后开始回升,96h时继续回升。 7、两组HELF Mcm2 mRNA、Mcm8 mRNA表达水平: 12h、24h、48h、72h、96h时两组HELF Mcm2 mRNA、Mcm8mRNA表达水平差异均有统计学意义(P＜0.05),12h和24h时感染组表达水平低于模拟感染组,48h、72h、96h时感染组表达水平明显高于模拟感染组。模拟感染组Mcm2 mRNA、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,较模拟感染组有明显滞后。 8、两组HELF Geminin mRNA表达水平: 12h、24h、48h和72h时两组HELF Geminin mRNA表达水平差异均有统计学意义(P＜0.05),12h和24h时感染组较模拟感染组明显增加,48h和72h时感染组较模拟感染组明显降低。感染组在感染后12h时Geminin mRNA表达水平最高,模拟感染组在感染后48h达最高峰。 结论 1、人巨细胞病毒感染人胚肺成纤维细胞后在其细胞核内进行复制、增殖。 2、人巨细胞病毒感染阻滞了人胚肺成纤维细胞周期,使大部分细胞停滞在G_1期。 3、人巨细胞病毒IE_172蛋白的表达提示人巨细胞病毒的早期感染。 4、人巨细胞病毒感染使人胚肺成纤维细胞复制前复合物主要成份Cdt1 mRNA、Mcm2 mRNA、Mcm8 mRNA的表达水平迟滞、Cdt1蛋白表达延迟。Cdc6 mRNA高水平表达。 5、人巨细胞病毒感染能诱导人胚肺成纤维细胞复制抑制因子Geminin异常表达,使Geminin/pre-RC平衡失调,阻止复制前复合物的形成,DNA合成障碍。这可能是人巨细胞病毒感染导致人胚肺成纤维细胞周期阻滞于G_1期的机制。
[Abstract]:objective
Objective to study the effects of human cytomegalovirus (HCMV) infection on the cell cycle and the replication complex (pre- replication complex, pre-) of human embryonic lung fibroblast (human embryonic lung fibroblast), and to explore the pathogenesis of infection.
Method
1, serum starvation synchronization HELF in G_0/G_1 phase, with the synchronization of HCMV infection as HELF infection group (cell number =1 * 10~6), and simulated infection group (cell number =1 * 10~6). Two groups were observed by the inverted microscope cell morphological changes to 7 days after infection, infection after 96 hours the cells were harvested and 12,24,48,72.
2, HELF cell cycle was detected by flow cytometry.
3, the level of HCMV IE_172 protein and Cdt1 protein in the nucleus of HELF was detected by immunocytochemical method.
4, the total RNA was extracted and semi quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression level of HELF complex, Cdt1 mRNA, Cdc6 mRNA, Mcm2 mRNA and Mcm8 mRNA in the two groups.
5, RT-PCR was used to detect the expression level of the two groups of HELF replicating inhibitor Geminin.
Result
1, two groups of HELF cell morphological changes:
Infection group HELF infection 72h after that, local cells became round and expanded, cell body and nucleus were huge. 7 days, all cells were found to be lesions. There was no morphological change in HELF at each time point in the simulated infection group.
2, two groups of HELF HCMV IE_172 protein positive products were expressed as follows:
The expression of HCMV IE_172 protein appeared in the nuclei of infected group HELF at 12h after infection, and there was no HCMV IE_172 protein positive product at all time points at HELF in the simulated infection group.
3, two groups of HELF cell cycle detection results:
In the G_1 infection group in 12h after infection of 70.4% cells, 24h 69.9% cells in G_1 phase; the mock infection group in 65.7% 12h after infection of the cells in the G_1 phase, 24h 43.8% cells in the G_1 phase. HELF in G_1 infection group stagnation infection group increased significantly compared with that of, the two groups, the differences were statistically significant (P < 0.01).
4, two groups of HELF Cdt1 mRNA expression levels:
The mock infection group after infection of 12h Cdt1 mRNA expression level is the highest, then gradually decreased to the lowest level at 72h, 96h rebounded slightly. The infection group after infection 12h Cdt1 expression level of mRNA is low, 24h increased slightly, 48h reached the peak and began to decline at 72h, 96h roughly maintain 72h level. 12h comparison, two groups had significantly different Cdt1 mRNA expression of 24h and 48h (P < 0.05), 12h and 24h infection group compared with the mock infection group decreased significantly, 48h infection group compared with mock infection group increased significantly. Simulated infection group Cdt1 mRNA level of the peak at 12h after infection, and the infection group reached a peak at 48h after infection, compared with the mock infection group was significantly delayed.
5, two groups of HELF Cdt1 protein levels:
12h, 24h, 96h and HELF Cdt1 protein levels in the two groups differences were statistically significant (P < 0.05), 48h, the difference was not statistically significant 72h HELF Cdt1 protein levels in the two groups (P > 0.05).12h and 96h infection group compared with the mock infection group decreased significantly, 24h infection group compared with the mock infection group obviously increased. The mock infection group after infection of 12h Cdt1 protein expression level is the highest, then gradually decreased to the lowest level at 24h, 96h rebounded slightly after infection of 12h infection group; the expression of Cdt1 protein level is low, reached a peak of 24h, 48h declined sharply, maintained a stable level low.
6, two groups of HELF Cdc6 mRNA expression levels:
12h, 24h, 48h level showed significant differences in two groups of HELF Cdc6 mRNA expression of 72h and 96h (P < 0.05), 12h, 24h and 48h infection group compared with the mock infection group was significantly increased, 72h and 96h simulated infection group than in infected group increased significantly. The infection group after infection of 12h Cdc6 mRNA the highest expression level, then decreased gradually, to the lowest level at 72h, 96h rebounded slightly; in the simulation when infected with 12h, the expression level of mRNA Cdc6 decreased gradually after the highest infection group, 48h, to the lowest level at 72h, and began to rise, 96h continues to rise.
7, two groups of HELF Mcm2 mRNA, Mcm8 mRNA expression level:
12h, 24h, 48h, 72h, 96h of the two groups of HELF Mcm2 mRNA, Mcm8mRNA expression levels were statistically significant difference (P < 0.05), 12h and 24h infection group expression level is lower than the mock infection group, 48h, 72h, 96h infection group was significantly higher than that simulated infection group. Simulated infection group Mcm2 mRNA. The expression of Mcm8 mRNA peaked at 24h after infection, and the infection group, the expression levels reached a peak at 48h after infection, compared with mock infection group had a significant lag.
8, two groups of HELF Geminin mRNA expression levels:
12h, 24h levels were statistically significant difference of two groups of HELF Geminin mRNA expression of 48h and 72h (P < 0.05), 12h and 24h infection group compared with the mock infection group was significantly increased, 48h and 72h infection group compared with mock infection group decreased significantly. The infection group after infection of 12h Geminin mRNA the highest expression level. The mock infection group at 48h after infection and reached the peak.
conclusion
1, human cytomegalovirus (HCMV) is infected with human embryonic lung fibroblasts and replicates and proliferate in its nucleus.
2, human cytomegalovirus infection blocks the cycle of human embryonic lung fibroblasts, which makes most of the cells stagnate in the G_1 phase.
3, the expression of human cytomegalovirus IE_172 protein suggests early infection of human cytomegalovirus (CCMV).
4, human cytomegalovirus infection causes the expression level of Cdt1 mRNA, Mcm2 mRNA, Mcm8 mRNA in human embryonic lung fibroblast. The expression level of Cdt1 is delayed, and the expression level of Cdt1 is delayed by.Cdc6 mRNA.
5, human cytomegalovirus infection to human embryonic lung fibroblast replication inhibitor induced abnormal expression of Geminin, Geminin/pre-RC balance, prevent the formation of pre replication complex, disorder of DNA synthesis. This may be the mechanism of human cytomegalovirus infection in human embryonic lung fibroblast cell cycle arrest in G_1 phase.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R373

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