人疱疹病毒6型感染对HSB2细胞周期和细胞凋亡的影响及机制研究

发布时间：2018-03-15 05:26

本文选题：人疱疹病毒6型　切入点：细胞增殖　出处：《南京医科大学》2010年博士论文　论文类型：学位论文

【摘要】：人类疱疹病毒6型(human herpesvirus 6, HHV-6)属于疱疹病毒β亚科,分为HHV-6A和B两个亚型。HHV-6是引起婴幼儿急疹(exanthem subitum, ES)的病原。文献报道HHV-6与淋巴系统增生性疾病、多发性硬化症(multiple sclerosis, MS)、急性脑炎、慢性疲劳综合症、病毒性心肌炎、器官移植后感染等多种疾病有关。HHV-6感染能影响宿主细胞周期进程甚至能诱导细胞凋亡,但其具体的分子机制目前还未进行过详细的研究。本课题以人T淋巴细胞系HSB2为研究对象,探讨HHV-6感染对HSB2细胞周期进程及细胞凋亡的影响及其分子机制。首先,我们大量收集HHV-6 GS标准株感染的CBMC细胞,反复冻融后,高速离心对病毒浓缩纯化。然后构建标准质粒,制作标准曲线,用实时荧光定量PCR的方法测定病毒滴度。其次,分别用细胞计数法和MTT法测定HHV-6感染对HSB2细胞增殖的影响,结果显示HHV-6感染能显著抑制细胞增殖,其作用具有时间依赖性;流式细胞术结果显示,感染细胞发生了G2/M期阻滞,进一步检测M期的标志分子磷酸化H3(Ser10),证明细胞周期停滞于G2期;实时荧光定量RT-PCR和Western blot结果显示,HHV-6感染使cyclinA2、cyclinB1、cyclinE1的蛋白水平和mRNA水平提高,cyclinD1的蛋白水平和mRNA水平均无明显变化;此外,Western blot和实时荧光定量PCR研究结果表明G2/M期细胞环境有利于病毒DNA复制和蛋白的表达。再次,本研究探讨了HHV-6诱导细胞G2阻滞的机制。体外激酶活性分析表明HHV-6感染抑制了cyclinB1-Cdk1(cdc2)激酶活性;Western blot结果显示磷酸化cdc2(Tyr15)表达量增加,总cdc2蛋白的表达也稍有增加;进一步研究表明HHV-6感染上调Weel蛋白表达,提高了Myt1激酶活性,并降低磷酸酶Cdc25C的活性。病毒感染使p53、p21、p27的蛋白和mRNA表达水平显著增加,并呈时间依赖性,并且磷酸化p53(Ser15)的表达也急剧增加;此外,HHV-6感染使磷酸化Chk2 (Thr68)和其下游分子磷酸化Cdc25C(Ser216)表达增加。以上结果提示,HHV-6感染可通过多途径,多分子协同诱导细胞G2阻滞。另外,在本研究中发现HHV-6感染导致G2期阻滞之后诱导了细胞凋亡,因此我们对其诱导凋亡的途径进行了探讨。流式细胞术PI单标法和Annexin V-FITC/PI双标检测均表明HHV-6在感染晚期可诱导凋亡。电子显微镜观察显示感染细胞染色质高度凝集、边缘化,细胞核碎裂呈碎片状,线粒体形态也发生了变化,表现为体积增大,肿胀,嵴紊乱、断裂; HHV-6感染后caspase-3,caspase-9活性显著升高,而caspase-8活性无变化;线粒体膜电位下降,Bcl-2蛋白表达水平下降,Bax表达水平增加。以上表明线粒体凋亡途径参与HHV-6诱导的细胞凋亡。综上所述,本研究结果提示HHV-6是通过多条途径、多种因子协同促进细胞的G2期阻滞,通过caspase依赖的线粒体途径诱导细胞凋亡。本研究为HHV-6感染相关疾病寻找新的治疗靶点提供了理论依据和新思路。
[Abstract]:Human herpesvirus 6 (HHV-6) belongs to herpesvirus 尾 subfamily. HHV-6 is the pathogen of infantile rash exanthem subtype (ES6). It is reported that HHV-6 and lymphoproliferative diseases, multiple sclerosis, multiple sclerosis, MSS, acute encephalitis. Chronic fatigue syndrome, viral myocarditis, infection after organ transplantation and other diseases. HHV-6 infection can affect the host cell cycle process and even induce cell apoptosis. However, the specific molecular mechanism has not been studied in detail. In this study, the effects of HHV-6 infection on the cell cycle progression and apoptosis of HSB2 cells and its molecular mechanism were studied. Firstly, we collected a large number of CBMC cells infected by HHV-6 GS standard strain. After freeze-thaw repeatedly, the virus was concentrated and purified by high speed centrifugation. Then the standard plasmid was constructed, the standard curve was made, and the virus titer was determined by real-time fluorescence quantitative PCR. Secondly, the effects of HHV-6 infection on the proliferation of HSB2 cells were determined by cell counting and MTT methods, respectively. The results showed that HHV-6 infection could significantly inhibit the proliferation of HSB2 cells in a time dependent manner, and flow cytometry showed that, The G _ 2 / M phase arrest occurred in infected cells, and the phosphorylation of H _ 3O _ (10), a marker of M phase, was further detected, which proved that the cell cycle stagnated at G _ 2 phase. The results of real-time fluorescence quantitative RT-PCR and Western blot showed that HHV-6 infection did not increase the protein level and mRNA level of cyclin D1 and mRNA. In addition, the results of Western blot and real-time fluorescent quantitative PCR showed that the G _ 2 / M phase cell environment was conducive to the replication of DNA and protein expression of the virus. Thirdly, we investigated the mechanism of G2 arrest induced by HHV-6. In vitro kinase activity analysis showed that HHV-6 infection inhibited cyclinB1-Cdk1cdc2) kinase activity. Western blot showed that phosphorylated cdc2mTyr15) expression was increased and the expression of total cdc2 protein was slightly increased. Further studies showed that HHV-6 infection upregulated the expression of Weel protein, increased the activity of Myt1 kinase and decreased the activity of phosphatase Cdc25C. Moreover, the expression of phosphorylated Chk2 Thr68) and its downstream phosphorylated Cdc25CmSer216) were increased by HHV-6 infection. These results suggest that HHV-6 infection can induce cell G2 arrest through multiple pathways and multimolecular co-induction. In addition, we found that HHV-6 infection induced apoptosis after G2 arrest. Flow cytometry and Annexin V-FITC / Pi double labeling showed that HHV-6 could induce apoptosis in the late stage of infection. Electron microscopy showed that the chromatin of infected cells was highly agglutinated and marginalized. After HHV-6 infection, the activity of caspase-3 and caspase-9 increased significantly, but the activity of caspase-8 did not change. The decrease of mitochondrial membrane potential decreased the expression of Bcl-2 protein and increased the expression of Bax, which suggested that the mitochondrial apoptosis pathway was involved in the apoptosis induced by HHV-6. To sum up, the results of this study suggest that HHV-6 promotes cell arrest in G2 phase through multiple pathways and multiple factors. Apoptosis was induced by caspase dependent mitochondrial pathway. This study provides a theoretical basis and a new idea for finding new therapeutic targets for HHV-6 infection related diseases.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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