本文選題：乙型肝炎 + HepG2細(xì)胞��； 參考：《重慶醫(yī)科大學(xué)》2011年碩士論文

【摘要】：乙型病毒性肝炎是全球公共衛(wèi)生問(wèn)題,全世界約有3.5億人因乙型肝炎病毒(Hepatitis B virus; HBV)導(dǎo)致慢性感染,慢性感染可能會(huì)發(fā)展為肝硬化甚至肝癌( hepatocarcinoma; HCC)。目前對(duì)慢性乙肝患者治療常用的藥物為I型干擾素(Interferon,IFNs)與核苷類(lèi)似物(nucleotide analogs,NAs),它們?cè)谂R床上得到了廣泛應(yīng)用并且在一定程度上減緩了疾病的惡化。但是由于HBV病毒變異速度快,藥物療效有限,還無(wú)法長(zhǎng)期有效地遏制HBV對(duì)人肝組織細(xì)胞的感染及其在胞內(nèi)DNA的復(fù)制。為了尋找新型治療方法及抗病毒藥物,需要深入了解HBV的感染機(jī)制,這就有待于篩選理想的HBV感染模型。近二十年來(lái)HBV體內(nèi)感染模型研究進(jìn)展緩慢,而HBV體外感染模型的研究取得了長(zhǎng)足的進(jìn)展,其中穩(wěn)定HBV表達(dá)細(xì)胞系由于其表達(dá)水平較穩(wěn)定且?guī)缀跛屑?xì)胞都能產(chǎn)生HBV,逐漸成為國(guó)內(nèi)外學(xué)者研究的熱點(diǎn)。 本研究通過(guò)構(gòu)建包含HBV1.1倍體及巨噬細(xì)胞病毒(cytomegalovirus;CMV)啟動(dòng)子的重組質(zhì)粒pneo-CH9/3091,使其表達(dá)新霉素(neomycin)抗性并將其穩(wěn)定轉(zhuǎn)染至人肝癌細(xì)胞HepG2,經(jīng)G418篩選,得到單克隆細(xì)胞株。通過(guò)多種分子生物學(xué)手段,如酶聯(lián)免疫吸附法(ELISA)檢測(cè)培養(yǎng)上清表面抗原(HBsAg)及e抗原(HBeAg)的分泌,Western blot檢測(cè)細(xì)胞內(nèi)HBV核心蛋白(HBcAg)的表達(dá),以及Southern blot檢測(cè)細(xì)胞內(nèi)核心顆粒HBV DNA的復(fù)制情況等,建立了一株HBV穩(wěn)定整合細(xì)胞株HepG2-H7。最后將此細(xì)胞株與HepG2.2.15細(xì)胞在HBV DNA的復(fù)制與蛋白表達(dá)水平等方面進(jìn)行了比較。本研究結(jié)果提示HepG2-H7細(xì)胞株有望應(yīng)用于抗病毒藥物的篩選和HBV感染機(jī)制的研究。
[Abstract]:Hepatitis B is a global public health problem. There are about 350 million people in the world who suffer from chronic infection caused by hepatitis B virus (HBV), which may develop into liver cirrhosis or even hepatocellular carcinoma (HCC). At present, the commonly used drugs in the treatment of chronic hepatitis B patients are interferon I (IFNs) and nucleotide analogs (nucleotide analogs), which have been widely used in clinic and slow down the deterioration of the disease to a certain extent. However, due to the rapid mutation rate of HBV virus and the limited efficacy of drugs, HBV infection of human liver tissue cells and its replication in cellular DNA can not be effectively controlled for a long time. In order to find new treatment methods and antiviral drugs, it is necessary to deeply understand the mechanism of HBV infection, which requires the screening of ideal HBV infection models. In the past two decades, the study of HBV in vivo infection model has been slow, while the study of HBV in vitro infection model has made great progress. Among them, stable HBV expression cell lines have become the focus of domestic and foreign scholars because of their stable expression level and the ability of almost all cells to produce HBV. In this study, a recombinant plasmid pneo-CH9 / 3091 containing HBV 1.1 ploidy and cytomegalovirus virus promoter was constructed and transfected stably into human hepatoma cell line HepG2. Monoclonal cell lines were obtained by G418 screening. The expression of HBV core protein (HBcAg) was detected by enzyme linked immunosorbent assay (Elisa) in culture supernatant surface antigen (HBsAg) and e antigen (HBeAg) by Western blot. A stable integrated HBV cell line HepG2-H7 was established by Southern blot. Finally, the replication and protein expression of HBV DNA were compared between this cell line and HepG2.2.15 cell line. The results suggest that HepG2-H7 cell line may be used in the screening of antiviral drugs and the mechanism of HBV infection.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R346

【參考文獻(xiàn)】

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