【作者】 李磊；

【导师】 杨东亮；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 目的：1．构建中国地区流行的B基因型HBV可复制性克隆，验证其在体内和体外的复制能力，为HBV复制相关研究及建立细胞和动物模型奠定基础。2．将具有良好复制能力的B基因型中国株HBV可复制性克隆亚克隆到腺相关病毒载体内，建立HBV慢性感染小鼠模型，同时建立A基因型HBV慢性感染小鼠模型。3．通过高压尾静脉注射的方法将逆转录病毒和非病毒shRNA表达载体导入到小鼠体内，比较其抗HBV的时效，以期获得较长期的HBV抑制效果。方法：1．以一例中国HBV B基因型感染患者来源的全基因克隆为母板，使用SapⅠ将HBV基因组切下后在体外自身环化。使用环化后的基因组为模板分两段分别扩增HBV1.3倍基因组片段，依次将其连接到克隆载体pBluescriptⅡKS(+)上获得含HBV1.3基因组的克隆。通过转染Huh7细胞系和高压尾静脉注射BAL B／cJ小鼠建立体外和体内HBV复制模型，采用ELISA、Southern Blot、Northern Blot以及免疫组织化学染色等方法检测HBV抗原分泌、复制中间体、转录子、抗原肝细胞内表达等情况，评价构建克隆的复制能力。2．将复制能力良好的HBV1.3倍基因组亚克隆到腺相关病毒载体pAAV-MCS内构建pAAV-HBV1.3质粒，体外转染Huh7细胞，检测其复制能力。采用高压尾静脉注射的方式将pAAV-HBV1.3克隆导入到C57 BL／6小鼠肝脏内，定期采血、取肝组织标本通过ELISA、Southern Blot、RT-PCR以及免疫组织化学染色等方法检测HBV抗原分泌、抗体产生、复制中间体、转录子、抗原肝细胞内表达等情况。同时使用Huang LR等报导的pAAV-HBV1.2克隆高压注射以建立A基因型的慢性感染小鼠模型。3．根据所构建的B基因型HBV可复制性克隆X区序列设计RNAi靶点，构建shRNA表达逆转录病毒载体，同pAAV-HBV1.3体外共转染Huh7细胞，筛选对HBV复制抑制效果较好的干扰靶点shRNA表达载体，并据此构建非病毒shRNA表达载体。将逆转录病毒及非病毒shRNA表达载体高压尾静脉注射导入小鼠肝脏，观察其抗HBV效果。结果：1．所构建的HBV1.3倍基因组克隆在Huh7细胞内和在BAL B／cJ小鼠可以正常分泌HBsAg和HBeAg，检测到复制中间体和转录子的产生。小鼠血清中可以检测到高滴度HBV DNA并随注射时间出现动态变化，小鼠肝细胞内可以检出呈胞浆型和胞膜型表达的HBcAg。2．pAAV-HBV1.3和AAV-HBV1.2两种克隆均可在C57 BL／6小鼠体内形成慢性HBV复制状态。在注射后的252天(A)／340天(QS)仍可在血清HBsAg阳性小鼠肝组织内检测到HBV复制中间体及HBV转录子的存在，肝细胞内有HBcAg表达，而HBsAg阴性的小鼠则无法检出。小鼠血清HBV DNA含量可维持在10~5-10~7 copies／mL水平，A组血清HBV DNA含量较QS组约高0.6-1.6log。但病毒血症持续时间不及QS组长，慢性化比例也低于QS组，注射24周时A组阳性率14.3％；QS组44.4％。3．非病毒shRNA表达载体干预组小鼠血清HBsAg阳性率、HBsAg含量、血清病毒滴度的有效抑制时间为1周以内。逆转录病毒shRNA表达载体干预组HBV抑制时间可持续4-6周，HBsAg阳性率在注射后前4周均保持在较低水平(18.2-27.3％)，HBsAg含量下降第2周仍可达81.0％，血清病毒滴度下降1.619-2.758log，小鼠肝组织内HBcAg阳性细胞数目明显减少。两组的抑制效果均随着注射时间的延长而逐渐下降。结论：1．成功构建了B基因型HBV可复制性克隆，在体内外均具有良好的复制能力。2．建立了B基因型和A基因型的HBV慢性感染小鼠模型。3．直接高压尾静脉注射逆转录病毒shRNA表达载体可以有效延长RNAi作用时间，有效抑制时间可持续至少4周。本课题的创新点：1．首次构建了同我国流行情况相符合的B基因型HBV可复制性克隆，并建立了HBV慢性感染小鼠模型。2．证实通过高压尾静脉注射方式导入逆转录病毒shRNA载体可以有效抑制HBV复制持续达4周以上。本课题研究的意义：1．构建B基因型HBV可复制性克隆为研究B基因型HBV相关病毒变异、复制能力等HBV生物学研究提供了良好的基础。2．建立的B基因型和A基因型的慢性HBV感染小鼠模型，具有易获取、制备简单、费用低廉、具有正常的机体免疫功能等优点。为研究抗病毒治疗、HBV复制机制、HBV感染慢性化机制、免疫发病机制、免疫调节等基础和应用研究提供了良好的小动物模型。3．高压尾静脉直接将逆转录病毒shRNA载体导入体内即可达到较长期抑制靶基因表达，具有无需包装病毒、安全、有效、时程长、操作简便等优点。为特定基因功能研究、导入特定外源基因至体内表达、肝脏靶向性治疗等方面研究奠定基础。更多还原

【Abstract】 Objects:1. In order to get a HBV replication competent clone of B genotype which is epidemic in Chinese, a plasmid that contain HBV 1.3 copies genome was constructed. It can provide a useful tool to study HBV biology, establish HBV replication cell model and animal model.2. Based on the genotype B HBV repliation competent clone, the HBV 1.3 copies genome was subcloned to a adenovirus associated virus vector, pAAV-MCS, expecting to establish a immunocompetent HBV chronically infected murine model by hydrodynamic injection the pAAV-HBV1.3 plasmid into tail vein. Meanwhile, pAAV-HBV1.2 plasmid which Huang LR had established genotype A HBV chronic infection in mice was employed as control.3. The shRNA expression retroviral vector and nonviral vector which targeting HBV X gene were hydrodynamic injected into murine tail vein directly, to verify if retroviral vector based RNAi could supress HBV replication in longer period. So it gived some light on the study method about specific gene’s function in a simple way.Methods: 1. The two corresponding sequences of HBV genome were amplified separately, than the target sequences were ligated in vitro. After the plasmid was verified by enzyme digestion, it was transfected into Huh7 cell line. The HBsAg and HBeAg concentration in culture supernatant were quantified. The transcript and replication intermediate of HBV were detected by Northern Blot and Southern Blot separately. On the other hand, the plasmid was hydrodynamic injected into BALB/cJ mice. Then the sera HBV DNA was quantified by real-time PCR, HBcAg in liver tissue was detected by immunohistochemistry staining.2. The HBV 1.3 copies genome DNA sequence was ampified by PCR, Then the PCR product was digested by Not I restriction enzyme and subcloned into pAAV-MCS vector. It was verified to be replicable by transfecting Huh7 cell in vitro. Then the pAAV- HBV1.3 plasmid was hydrodynamic injected into tail vein of C57 BL/6 mice. Mice sera and liver tissues were collected at indicated time point in the following 48 weeks or more. The samples were detected by ELISA, southern blot, real-time quantified PCR, reverse transcriptional PCR and et al. At the same time, the pAAV-HBV1.2 plasmid, which Huang et al used to establish HBV chronic infection mice model, was employed in parallel.3. The HBV X gene specific RNA interference target sequences were desigened. Then the corresponding shRNA expression retroviral vectors were constructed. These vectors were cotransfected into Huh7 cell line with pAAV-HBV1.3 plasmid we constructed and the best RNAi target expression vector was selected. The HBx 285nt specific shRNA expression nonviral vector was also constructed. Both retroviral and noviral HBx285 shRNA expression vectors were injected into C57 BL/6 mice with same quantity pAAV-HBV1.3 plasmid to study if retroviral vector in this transduction way can be functional in a longer period than nonviral vector. Results:1. After be transfected into Huh7 cell line, the HBsAg and HBeAg can be secreted in culture supernatant, and the transcript and replication intermediate of HBV also can be detected. The HBV DNA was detected with high titer in mice sera and HBcAg was expressed in hepatocytes of mice.2. Both pAAV-HBV1.3 and pAAV-HBV1.2 plasmid can be used to establish HBV chronic infection in mice. The pAAV-HBV1.2 treated mice can sustain sera HBsAg postive at least 48 weeks. The HBsAg can be postive until 36 weeks postinjection in mice hydrodynamic injected with pAAV-HBV1.3 we constructed, but the viral load was higher than the pAAV-HBV1.2 mice in 0.6-1.6log value, sustaining in 10~5-10~7copies/mL level. The HBsAg postive percentage were 14.3% and 44.4% in pAAV-HBV1.3 and pAAV-HBV1.2 injected mice at 24 weeks postinjection. The serum HBsAg positive sustaining time of pAAV-HBV1.2 injected mice was longer than pAAV-HBV1.3 counterparts.3. The retroviral vector can enlong its shRNA antiviral effect time than nonviral vector. Sera HBsAg positive percentage, HBsAg quantity, serum HBV DNA concentration and HBcAg positive hepatocytes number can lowered significantly at least 4 weeks after retroviral vector injected mice versus about almost 1 week in nonviral vector mice.Conclusions:1. The replication competent HBV clone of genotype B we constructed can replicate in vitro and in vivo.2. The genotype B and A HBV chronically infected murine models are successfully established.3. The retroviral shRNA expression vector can prolong the time of HBV replication supression by RNAi in hydrodynamic tail vein injection.更多还原