本文選題：乙型感染病毒(HBV)　切入點(diǎn)：短發(fā)夾RNA(shRNA)　出處：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景：乙型肝炎病毒引起的慢性乙型肝炎是一種重大的傳染性疾病。乙型肝炎主要流行于中國(guó)及其它一些亞洲國(guó)家,目前約有十分之一的中國(guó)人口是乙型肝炎病毒攜帶者。慢性乙型肝炎可以導(dǎo)致肝硬化及肝癌的發(fā)生,并且目前沒(méi)有藥物可以完全根治,治療種類少且療效有限。因此,尋找一種有效的治療乙型肝炎的方法迫在眉睫。RNA干擾(RNA interfering)是指一些小的雙鏈RNA (dsRNA)可高效、特異的促使mRNA降解,阻斷相應(yīng)基因表達(dá)。應(yīng)用RNAi治療乙型肝炎是一種有效的治療策略,能夠突破普通藥物治療療效的局限性,克服易產(chǎn)生逃逸突變株使病情更加惡化等缺點(diǎn)。但化學(xué)合成的siRNA成本高、不穩(wěn)定、體內(nèi)傳輸困難,進(jìn)入細(xì)胞后易被降解,因此,需要研發(fā)一種新的傳輸方式,病毒載體為我們提供了一個(gè)契機(jī)。腺相關(guān)病毒AAV(Adeno associated virus)屬于人類細(xì)小病毒科(parvoviridae)。白建立第一個(gè)AAV的感染性克隆以來(lái),重組AAV載體被廣泛應(yīng)用到基因治療研究及臨床前/臨床實(shí)驗(yàn)中,嘗試治療多種疾病。AAV具有無(wú)致病性、低免疫原性、能轉(zhuǎn)導(dǎo)分裂和非分裂的細(xì)胞、可介導(dǎo)外源基因長(zhǎng)期表達(dá)等優(yōu)點(diǎn)。因此,本課題選擇腺相關(guān)病毒作為短發(fā)夾結(jié)構(gòu)RNA(shRNA)傳遞的載體,進(jìn)行抗乙型肝炎病毒的研究。乙型肝炎病毒宿主范圍窄,且沒(méi)有適合的動(dòng)物模型進(jìn)行抗病毒藥物的評(píng)估,嚴(yán)重制約了抗乙型肝炎病毒藥物的研發(fā)。因此研發(fā)一種新型的乙型肝炎病毒持續(xù)性感染小鼠模型對(duì)研究慢性乙型肝炎的致病機(jī)制及新型抗病毒藥物的篩查和評(píng)估具有重要意義。研究目的：1、構(gòu)建乙型肝炎病毒持續(xù)性感染小鼠模型。2、構(gòu)建一系列AAV2-shRNA表達(dá)載體,體外驗(yàn)證各AAV2-shRNA的抗病毒效果。3、在乙型肝炎病毒持續(xù)性感染小鼠模型上評(píng)估AAV2-shRNA的抗病毒效果。實(shí)驗(yàn)方法：1、將可復(fù)制性的1.2拷貝的HBV克隆到去除rep和cap基因的腺相關(guān)病毒質(zhì)粒表達(dá)載體pSSV9 (rep-/cap-)中,構(gòu)建出質(zhì)粒表達(dá)載體pSSV9-1.2HBV,并利用三質(zhì)粒共轉(zhuǎn)染技術(shù)制備成重組病毒(AAV8-1.2HBV)。將AAV8-1.2HBV尾靜脈注射C57BL/6小鼠,定期采血、取組織樣本通過(guò)qPCR、RT-qPCR、ELISA、免疫組織化學(xué)染色、Masson染色等方法檢測(cè)血清中HBV DNA變化、肝組織中HBV cDNA變化、血清中抗原分泌情況、肝細(xì)胞內(nèi)抗原表達(dá)情況、病理變化及模型小鼠肝組織纖維化指標(biāo)的變化等。2、制備一系列shRNA質(zhì)粒表達(dá)載體pSC-H1-shRNA和串聯(lián)形式的shRNA質(zhì)粒表達(dá)載體pSC-H1-shRNAmultiple,利用三質(zhì)粒共轉(zhuǎn)染技術(shù)包裝成AAV2-shRNA重組病毒。用AAV2-shRNA重組病毒感染HepG2.2.15細(xì)胞系,定期收獲培養(yǎng)上清液和培養(yǎng)細(xì)胞,通過(guò)qPCR、RT-qPCR和ELISA等方法從DNA水平,RNA水平和蛋白水平評(píng)估各AAV2-shRNA抗病毒效果。3、將體外篩選出的抗病毒效果顯著的AAV2-shRNA病毒尾靜脈注射乙型肝炎病毒持續(xù)性感染小鼠模型,定期采血、取組織樣木通過(guò)測(cè)量與方法1中相同的各項(xiàng)指標(biāo),評(píng)估AAV2-shRNA體內(nèi)抗HBV的效果。結(jié)果：1、模型小鼠血清中可持續(xù)檢測(cè)到高滴度的HBV DNA (107 copy/mL)超過(guò)6個(gè)月：模型小鼠血清和肝細(xì)胞中分別可檢測(cè)到HBsAg、HBeAg的分泌與HBsAg、 HBcAg的表達(dá)。病理切片及纖維化指標(biāo)定量檢測(cè)顯示,小鼠肝組織沒(méi)有明顯的急性炎癥反應(yīng),但是能誘導(dǎo)肝組織脂肪變性和纖維化。2、所有的AAV2-shRNA體外均有抑制HBV的能力,抗病毒效果呈劑量依賴關(guān)系,但是感染劑量超過(guò)MOI=104時(shí),抗HBV效果未有顯著增加。相同感染劑量下,AAV2-shRNA1+3對(duì)HBV的抑制率要顯著大于AAV2-shRNA1和AAV2-shRNA1+3+4。3、體內(nèi)驗(yàn)證AAV2-shRNA.1, AAV2-shRNA1+3抗HBV效果,各項(xiàng)結(jié)果顯示AAV2-shRNA1+3具有更強(qiáng)的抗病毒能力,與體外結(jié)果相符。檢測(cè)纖維化相關(guān)指標(biāo)顯示,AAV2-shRNA1+3進(jìn)行抗HBV治療可以改善肝組織脂肪變性和肝組織纖維化的程度。結(jié)論：1、成功構(gòu)建了乙型肝炎病毒持續(xù)性感染小鼠模型,此模型不產(chǎn)生明顯的急性炎癥反應(yīng),但是能誘導(dǎo)小鼠肝組織纖維化,為慢性乙型肝炎和肝硬化治療藥物的篩選和評(píng)估提供了一個(gè)良好的平臺(tái)。2、體外篩選出的AAV2-shRNA1+3,具有顯著的抗HBV復(fù)制的作用。3、AAV2-shRNA1+3在HBV持續(xù)性感染小鼠模型上具有顯著的抗病毒作用,能夠改善肝臟組織的脂肪變性和纖維化程度,是非常有前景的慢性乙型肝炎和肝硬化治療的候選藥物。
[Abstract]:Background: hepatitis B virus associated chronic hepatitis B is a serious infectious disease. Hepatitis B is popular mainly in the Chinese and some other Asian countries, there are about 1/10 of the population is China hepatitis B virus carriers. Chronic hepatitis B can lead to cirrhosis and liver cancer, and there is no drug can cure completely. Treatment of less species and limited efficacy. Therefore, to find an effective method for the treatment of hepatitis B.RNA (RNA interfering) imminent interference refers to some small double stranded RNA (dsRNA) can be highly effective, specific to the degradation of mRNA, blocking the expression of the corresponding gene. Application of RNAi for the treatment of hepatitis B is a kind of effective treatment strategies to break the limitation, curative effect of drug treatment, to overcome the shortcomings of escape mutants easily make the condition worse. But the high cost of chemical synthesis of siRNA, Unstable body transmission difficulties, after entering the cell susceptible to degradation, therefore, need to develop a new mode of transmission, virus vector provides an opportunity for us. Adeno-associated virus AAV (Adeno associated virus) belongs to the family of human parvovirus (Parvoviridae). The infectious clone of white since the establishment of the first AAV. The recombinant AAV vector has been widely applied to clinical trials and preclinical study of gene therapy in the treatment of various diseases / try,.AAV has no pathogenicity, low immunogenicity, can transduce dividing and non dividing cells, the advantages of exogenous gene mediated by long-term expression. Therefore, this paper choose the adeno-associated virus as short hairpin RNA (shRNA) transfer vector of anti hepatitis B virus. Hepatitis B virus host range is narrow, and there is no suitable animal model for the evaluation of antiviral drugs, which seriously restrict the anti hepatitis B virus Drug research and development. Therefore the research on the screening and evaluation of a new type of hepatitis B virus infection of mice model for chronic hepatitis B pathogenesis and new antiviral drugs has important significance. The purpose of the study: 1. Construction of hepatitis B virus persistent infection mouse model of.2, constructed a series of AAV2-shRNA expression vector in vitro to verify the the antiviral effect of.3 in AAV2-shRNA, infection mouse model to assess AAV2-shRNA sustained antiviral effect of hepatitis B virus. Methods: 1, will be copied 1.2 copies of HBV cloned into adeno-associated virus plasmid removal of rep and cap gene expression vector pSSV9 (rep-/cap-), constructed the plasmid expression vector pSSV9-1.2HBV, and using three plasmid co transfection techniques to prepare recombinant virus (AAV8-1.2HBV). The AAV8-1.2HBV injected into the tail vein of C57BL/6 mice, regular sampling, the tissue samples Through qPCR, RT-qPCR, ELISA, immunohistochemistry, HBV DNA staining method for detection of serum Masson changes, changes of HBV cDNA in liver tissue, serum antigen, antigen expression in liver cells, fibrosis and pathological changes of mice liver tissue of.2, preparation of a series of shRNA expression plasmid pSC-H1-shRNA vector and shRNA plasmid series form expression vector pSC-H1-shRNAmultiple were transfected into AAV2-shRNA packaging technology with three plasmids. The recombinant virus infected HepG2.2.15 cell line with AAV2-shRNA recombinant virus, regular harvest and culture supernatant of cultured cells, through qPCR, RT-qPCR and ELISA from the DNA level, the AAV2-shRNA level of RNA and.3 to evaluate the antiviral effect of protein the level will be screened in vitro antiviral effect of AAV2-shRNA virus intravenous injection of hepatitis B virus persistent infection of mice Type, regular sampling, the tissue sample wood and method by measuring the indexes of 1 in the same, to assess the effect of AAV2-shRNA in vivo of HBV. Results: 1. Serum HBV was detected in a mouse model of sustainable high titer of DNA (107 copy/mL) for more than 6 months: the model of mouse serum and liver cells were detected to HBsAg, HBsAg and secretion of HBeAg, HBcAg expression showed pathological sections and fibrosis. Quantitative detection of liver tissue of mice without obvious acute inflammatory reaction, but can induce hepatic steatosis and fibrosis in.2, AAV2-shRNA were all inhibited HBV in vitro, the antiviral effect was dose-dependent, but the infection dose more than MOI=104, no anti HBV effect will increase significantly. The same infection dose, AAV2-shRNA1+3 inhibition rate of HBV was significantly higher than that of AAV2-shRNA1 and AAV2-shRNA1+3+4.3 in vivo validation AAV2-shRNA.1, AAV2-shRN A1+3 anti HBV effect, the results show that AAV2-shRNA1+3 has stronger antiviral ability, consistent with in vitro results. Showed fibrosis related indexes, AAV2-shRNA1+3 anti HBV treatment can improve hepatic steatosis and the degree of hepatic fibrosis. Conclusion: 1. The successful construction of hepatitis B virus infection mouse model continues, this model does not produce acute inflammatory reaction obviously, but can induce liver fibrosis in mice, provide a good platform for.2 screening and evaluating drugs for the treatment of chronic hepatitis B and cirrhosis, in vitro screened AAV2-shRNA1+3, inhibit HBV replication.3 effect of AAV2-shRNA1+3 in persistent HBV infection mouse model with antiviral effect that can improve liver steatosis and fibrosis, chronic hepatitis B and liver cirrhosis is a very promising treatment A candidate drug for treatment.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.62;R-332

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