本文選題：慢病毒　切入點：X蛋白　出處：《福建醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目的:以慢病毒為載體構(gòu)建穩(wěn)定表達乙型肝炎病毒X基因(HBV X)的HL7702細(xì)胞株,探討HBV X穩(wěn)定表達對HL7702細(xì)胞線粒體結(jié)構(gòu)和功能的影響。方法:首先根據(jù)In-Fusion技術(shù)原理設(shè)計克隆引物,采用PCR技術(shù)從真核質(zhì)粒pc DNA3.1-X中擴增出含HBV X基因DNA全長片段,用In-Fusion交換酶將PCR產(chǎn)物與線性化慢病毒載體質(zhì)粒連接,經(jīng)PCR、測序、質(zhì)粒表達檢測鑒定。將構(gòu)建成功的重組慢病毒質(zhì)粒PLOV.CMV.e GFP.2A.3×FlagHBx.EF1a.Pura與包裝質(zhì)粒ps PAX2、包膜質(zhì)粒p MD2.G三質(zhì)粒瞬時共轉(zhuǎn)染293T細(xì)胞以包裝慢病毒顆粒,慢病毒顆粒的滴度采用熒光定量PCR的方法檢測。包裝好的重組慢病毒顆粒感染HL7702細(xì)胞,并經(jīng)過嘌呤霉素篩選獲得穩(wěn)定表達HBV X的細(xì)胞株HL7702/HBx,并經(jīng)過RT-PCR和Western-blot實驗證實HL7702/HBx細(xì)胞內(nèi)有HBV X穩(wěn)定持續(xù)的表達。以半定量RT-PCR方法和Western-blot方法檢測細(xì)胞色素C氧化酶III(COXIII)表達水平變化,酶動力學(xué)方法檢測線粒體細(xì)胞色素C氧化酶活性,流式細(xì)胞術(shù)檢測細(xì)胞內(nèi)ROS水平和線粒體跨膜電位,利用激光掃描共聚焦顯微鏡采集熒光信號觀察COXIII與HBV X蛋白(HBx)在肝細(xì)胞HL7702內(nèi)的定位,掃描電鏡下觀察HL7702/HBx細(xì)胞線粒體超微結(jié)構(gòu)的改變。結(jié)果:成功構(gòu)建表達3×FlagHBx融合基因的重組慢病毒表達載體,經(jīng)293T細(xì)胞包裝和濃縮后,重組慢病毒滴度為8.61×107IU/ml。慢病毒感染HL7702細(xì)胞后經(jīng)嘌呤霉素藥物篩選,得到穩(wěn)定表達HBx的新細(xì)胞株HL7702/HBx,經(jīng)RT-PCR和Western-blot分析結(jié)果顯示HL7702/HBx細(xì)胞能夠持續(xù)穩(wěn)定表達HBx。HBx在細(xì)胞漿和細(xì)胞核中表達,而細(xì)胞漿中HBx可分布于線粒體,并與COXIII共定位表達。掃描電鏡下發(fā)現(xiàn)穩(wěn)定表達HBx的細(xì)胞線粒體較空白對照組和空載對照組線粒體脊稍腫脹,細(xì)胞形態(tài)及結(jié)構(gòu)無明顯改變。半定量RT-PCR實驗結(jié)果發(fā)現(xiàn)COXIII m RNA表達水平無明顯改變,Western-blot實驗結(jié)果表明HL7702/HBx細(xì)胞內(nèi)COXIII蛋白表達上調(diào),線粒體細(xì)胞色素C氧化酶活性水平升高,同時也發(fā)現(xiàn)細(xì)胞內(nèi)ROS水平上升,而線粒體跨膜電位沒有發(fā)生明顯改變。結(jié)論:成功構(gòu)建穩(wěn)定表達HBx的細(xì)胞株HL7702/HBx。細(xì)胞漿中HBx可定位于線粒體并與COXIII相互作用,通過轉(zhuǎn)錄后水平調(diào)節(jié)COXIII表達,從而提高線粒體細(xì)胞色素C氧化酶活性和細(xì)胞內(nèi)ROS水平,導(dǎo)致線粒體脊稍腫脹,但不改變線粒體跨膜電位和細(xì)胞形態(tài)。
[Abstract]:Objective: to construct a HL7702 cell line expressing HBV X gene stably by lentivirus and to investigate the effect of stable expression of HBV X on the mitochondrial structure and function of HL7702 cells. Methods: firstly, primers were designed according to the principle of In-Fusion technique. The full-length DNA fragment containing HBV X gene was amplified by PCR from eukaryotic plasmid PC DNA3.1-X. The PCR product was ligated with the linearized lentivirus vector plasmid by In-Fusion exchange enzyme. The recombinant lentivirus plasmid PLOV.CMV.e GFP.2A.3 脳 FlagHBx.EF1a.Pura and the packaging plasmid PS PAX2 were constructed and cotransfected into 293T cells to package the lentivirus particles. The titer of lentivirus particles was detected by fluorescent quantitative PCR. Packaged recombinant lentivirus particles infected HL7702 cells. The cell line HL7702 / HBxexpressing HBV X stably was obtained by purine mycin screening, and the expression of HBV X was confirmed by RT-PCR and Western-blot experiments. The expression of HBV X was detected by semi-quantitative RT-PCR method and Western-blot method. The activity of cytochrome C oxidase in mitochondria was detected by enzyme kinetic method, the intracellular ROS level and mitochondrial transmembrane potential were detected by flow cytometry. Fluorescence signals were collected by laser scanning confocal microscope to observe the localization of COXIII and HBV X protein in HL7702 of hepatocytes. Results: the recombinant lentivirus expression vector expressing 3 脳 FlagHBx fusion gene was successfully constructed and packaged and concentrated by 293T cells. The recombinant lentivirus titer was 8.61 脳 10 ~ (7) IU / ml. After infection of lentivirus with HL7702 cells, a novel cell line HL7702 / HBxexpressing HBx was obtained by purine mycin drug screening. The results of RT-PCR and Western-blot analysis showed that HL7702/HBx cells could express HBx.HBx in cytoplasm and nucleus steadily. However, HBx in cytoplasm could be distributed in mitochondria and co-expressed with COXIII. Under scanning electron microscope, mitochondria with stable expression of HBx were slightly swollen than those of blank control group and no-load control group. The results of semi-quantitative RT-PCR assay showed that the expression of COXIII m RNA did not change significantly. The results of Western-blot analysis showed that the expression of COXIII protein was up-regulated and the activity of cytochrome C oxidase in mitochondria was increased in HL7702/HBx cells. At the same time, it was also found that the level of ROS increased, but the mitochondrial transmembrane potential did not change significantly. Conclusion: the stable expression of HBx in HL7702 / HBx. the HBx in cytoplasm can be located in mitochondria and interact with COXIII. The expression of COXIII was regulated at posttranscriptional level, which increased the activity of cytochrome C oxidase and the level of intracellular ROS, which resulted in a slight swelling of mitochondria ridges, but did not change the mitochondrial transmembrane potential and cell morphology.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R512.6;R735.7

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