本文選題：人皰疹病毒6型　切入點：細胞增殖　出處：《南京醫(yī)科大學》2010年博士論文　論文類型：學位論文

【摘要】：人類皰疹病毒6型(human herpesvirus 6, HHV-6)屬于皰疹病毒β亞科,分為HHV-6A和B兩個亞型。HHV-6是引起嬰幼兒急疹(exanthem subitum, ES)的病原。文獻報道HHV-6與淋巴系統(tǒng)增生性疾病、多發(fā)性硬化癥(multiple sclerosis, MS)、急性腦炎、慢性疲勞綜合癥、病毒性心肌炎、器官移植后感染等多種疾病有關(guān)。HHV-6感染能影響宿主細胞周期進程甚至能誘導細胞凋亡,但其具體的分子機制目前還未進行過詳細的研究。本課題以人T淋巴細胞系HSB2為研究對象,探討HHV-6感染對HSB2細胞周期進程及細胞凋亡的影響及其分子機制。 首先,我們大量收集HHV-6 GS標準株感染的CBMC細胞,反復凍融后,高速離心對病毒濃縮純化。然后構(gòu)建標準質(zhì)粒,制作標準曲線,用實時熒光定量PCR的方法測定病毒滴度。 其次,分別用細胞計數(shù)法和MTT法測定HHV-6感染對HSB2細胞增殖的影響,結(jié)果顯示HHV-6感染能顯著抑制細胞增殖,其作用具有時間依賴性;流式細胞術(shù)結(jié)果顯示,感染細胞發(fā)生了G2/M期阻滯,進一步檢測M期的標志分子磷酸化H3(Ser10),證明細胞周期停滯于G2期;實時熒光定量RT-PCR和Western blot結(jié)果顯示,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水平均無明顯變化;此外,Western blot和實時熒光定量PCR研究結(jié)果表明G2/M期細胞環(huán)境有利于病毒DNA復制和蛋白的表達。 再次,本研究探討了HHV-6誘導細胞G2阻滯的機制。體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結(jié)果顯示磷酸化cdc2(Tyr15)表達量增加,總cdc2蛋白的表達也稍有增加;進一步研究表明HHV-6感染上調(diào)Weel蛋白表達,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表達水平顯著增加,并呈時間依賴性,并且磷酸化p53(Ser15)的表達也急劇增加;此外,HHV-6感染使磷酸化Chk2 (Thr68)和其下游分子磷酸化Cdc25C(Ser216)表達增加。以上結(jié)果提示,HHV-6感染可通過多途徑,多分子協(xié)同誘導細胞G2阻滯。 另外,在本研究中發(fā)現(xiàn)HHV-6感染導致G2期阻滯之后誘導了細胞凋亡,因此我們對其誘導凋亡的途徑進行了探討。流式細胞術(shù)PI單標法和Annexin V-FITC/PI雙標檢測均表明HHV-6在感染晚期可誘導凋亡。電子顯微鏡觀察顯示感染細胞染色質(zhì)高度凝集、邊緣化,細胞核碎裂呈碎片狀,線粒體形態(tài)也發(fā)生了變化,表現(xiàn)為體積增大,腫脹,嵴紊亂、斷裂; HHV-6感染后caspase-3,caspase-9活性顯著升高,而caspase-8活性無變化;線粒體膜電位下降,Bcl-2蛋白表達水平下降,Bax表達水平增加。以上表明線粒體凋亡途徑參與HHV-6誘導的細胞凋亡。 綜上所述,本研究結(jié)果提示HHV-6是通過多條途徑、多種因子協(xié)同促進細胞的G2期阻滯,通過caspase依賴的線粒體途徑誘導細胞凋亡。本研究為HHV-6感染相關(guān)疾病尋找新的治療靶點提供了理論依據(jù)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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前3條

1 姚X,范萍,季曉輝,周瑤璽;人類皰疹病毒6型刺激人臍帶血單個核細胞產(chǎn)生α-干擾素的研究[J];上海免疫學雜志;1998年01期

2 Richard Y. ZHAO,Robert T. ELDER;Viral infections and cell cycle G2/M regulation[J];Cell Research;2005年03期

3 彭光勇,姚X,任強,季曉輝;人類皰疹病毒6、7型體外感染淋巴細胞對四種細胞因子分泌的影響[J];中華微生物學和免疫學雜志;2000年06期

，

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