AAV介导的HBV持续性感染和纤维化小鼠模型的建立及抗病毒药物评估

发布时间：2018-03-04 02:14

本文选题：乙型感染病毒(HBV)　切入点：短发夹RNA(shRNA)　出处：《北京协和医学院》2013年博士论文　论文类型：学位论文

【摘要】：研究背景：乙型肝炎病毒引起的慢性乙型肝炎是一种重大的传染性疾病。乙型肝炎主要流行于中国及其它一些亚洲国家,目前约有十分之一的中国人口是乙型肝炎病毒携带者。慢性乙型肝炎可以导致肝硬化及肝癌的发生,并且目前没有药物可以完全根治,治疗种类少且疗效有限。因此,寻找一种有效的治疗乙型肝炎的方法迫在眉睫。RNA干扰(RNA interfering)是指一些小的双链RNA (dsRNA)可高效、特异的促使mRNA降解,阻断相应基因表达。应用RNAi治疗乙型肝炎是一种有效的治疗策略,能够突破普通药物治疗疗效的局限性,克服易产生逃逸突变株使病情更加恶化等缺点。但化学合成的siRNA成本高、不稳定、体内传输困难,进入细胞后易被降解,因此,需要研发一种新的传输方式,病毒载体为我们提供了一个契机。腺相关病毒AAV(Adeno associated virus)属于人类细小病毒科(parvoviridae)。白建立第一个AAV的感染性克隆以来,重组AAV载体被广泛应用到基因治疗研究及临床前/临床实验中,尝试治疗多种疾病。AAV具有无致病性、低免疫原性、能转导分裂和非分裂的细胞、可介导外源基因长期表达等优点。因此,本课题选择腺相关病毒作为短发夹结构RNA(shRNA)传递的载体,进行抗乙型肝炎病毒的研究。乙型肝炎病毒宿主范围窄,且没有适合的动物模型进行抗病毒药物的评估,严重制约了抗乙型肝炎病毒药物的研发。因此研发一种新型的乙型肝炎病毒持续性感染小鼠模型对研究慢性乙型肝炎的致病机制及新型抗病毒药物的筛查和评估具有重要意义。研究目的：1、构建乙型肝炎病毒持续性感染小鼠模型。2、构建一系列AAV2-shRNA表达载体,体外验证各AAV2-shRNA的抗病毒效果。3、在乙型肝炎病毒持续性感染小鼠模型上评估AAV2-shRNA的抗病毒效果。实验方法：1、将可复制性的1.2拷贝的HBV克隆到去除rep和cap基因的腺相关病毒质粒表达载体pSSV9 (rep-/cap-)中,构建出质粒表达载体pSSV9-1.2HBV,并利用三质粒共转染技术制备成重组病毒(AAV8-1.2HBV)。将AAV8-1.2HBV尾静脉注射C57BL/6小鼠,定期采血、取组织样本通过qPCR、RT-qPCR、ELISA、免疫组织化学染色、Masson染色等方法检测血清中HBV DNA变化、肝组织中HBV cDNA变化、血清中抗原分泌情况、肝细胞内抗原表达情况、病理变化及模型小鼠肝组织纤维化指标的变化等。2、制备一系列shRNA质粒表达载体pSC-H1-shRNA和串联形式的shRNA质粒表达载体pSC-H1-shRNAmultiple,利用三质粒共转染技术包装成AAV2-shRNA重组病毒。用AAV2-shRNA重组病毒感染HepG2.2.15细胞系,定期收获培养上清液和培养细胞,通过qPCR、RT-qPCR和ELISA等方法从DNA水平,RNA水平和蛋白水平评估各AAV2-shRNA抗病毒效果。3、将体外筛选出的抗病毒效果显著的AAV2-shRNA病毒尾静脉注射乙型肝炎病毒持续性感染小鼠模型,定期采血、取组织样木通过测量与方法1中相同的各项指标,评估AAV2-shRNA体内抗HBV的效果。结果：1、模型小鼠血清中可持续检测到高滴度的HBV DNA (107 copy/mL)超过6个月：模型小鼠血清和肝细胞中分别可检测到HBsAg、HBeAg的分泌与HBsAg、 HBcAg的表达。病理切片及纤维化指标定量检测显示,小鼠肝组织没有明显的急性炎症反应,但是能诱导肝组织脂肪变性和纤维化。2、所有的AAV2-shRNA体外均有抑制HBV的能力,抗病毒效果呈剂量依赖关系,但是感染剂量超过MOI=104时,抗HBV效果未有显著增加。相同感染剂量下,AAV2-shRNA1+3对HBV的抑制率要显著大于AAV2-shRNA1和AAV2-shRNA1+3+4。3、体内验证AAV2-shRNA.1, AAV2-shRNA1+3抗HBV效果,各项结果显示AAV2-shRNA1+3具有更强的抗病毒能力,与体外结果相符。检测纤维化相关指标显示,AAV2-shRNA1+3进行抗HBV治疗可以改善肝组织脂肪变性和肝组织纤维化的程度。结论：1、成功构建了乙型肝炎病毒持续性感染小鼠模型,此模型不产生明显的急性炎症反应,但是能诱导小鼠肝组织纤维化,为慢性乙型肝炎和肝硬化治疗药物的筛选和评估提供了一个良好的平台。2、体外筛选出的AAV2-shRNA1+3,具有显著的抗HBV复制的作用。3、AAV2-shRNA1+3在HBV持续性感染小鼠模型上具有显著的抗病毒作用,能够改善肝脏组织的脂肪变性和纤维化程度,是非常有前景的慢性乙型肝炎和肝硬化治疗的候选药物。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62;R-332

【共引文献】