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建立基于高压水动力法的乙型肝炎病毒(HBV)转染小鼠模型,并进一步建立和优化乙肝动物模型研究方法。首先构建了含腺相关病毒倒转末端重复序列元件与包含1.3个拷贝HBV基因组(ayw亚型)的HBV表达质粒(pAAV-HBV1.3);并将pAAV-HBV1.3质粒经高压水动力法尾静脉注射C57BL/6小鼠,不同时间点采集血液和肝组织标本,ELISA检测血清HBsAg、HBeAg表达;Real-time PCR检测血清及肝组织病毒载量;HE染色、免疫组化染色检测肝组织病理学改变及病毒抗原在肝组织中的定位及表达;最后采用免疫抑制剂地塞米松注射液(DEX)腹腔注射小鼠,建立免疫功能抑制小鼠模型,在此基础上制备乙肝病毒转染小鼠模型,并进行血清HBsAg、HBeAg检测。结果是正常免疫状态下,小鼠转染pAAV-HBV1.3 10d时血清及肝组织HBV相关抗原阳性,30d后HBV相关抗原检测阴性,但30d和60d血清及肝组织病毒载量检测均为阳性,且与对照组差异显著(P<0.01,P<0.05);经地塞米松注射后处于免疫抑制状态下的高压水动力法建立的乙肝病毒转染小鼠,则在60d仍可检测到HBsAg、HBeAg的表达。以上结果表明通过高压水动力法建立了急性乙肝小鼠模型,通过抑制小鼠免疫状态,可延长病毒在小鼠体内存留时间。该模型建立为HBV疫苗评价、药物开发及乙肝相关致病机理研究奠定了基础。
A hepatitis B virus (HBV) -transfected mouse model based on high-pressure hydrodynamic method was established, and the research methods of animal model of hepatitis B were further established and optimized. First of all, the recombinant adenovirus vector containing pAAV-HBV1.3 was constructed by reverse transcription of the adeno-associated virus and the HBV expression plasmid (pAAV-HBV1.3) containing 1.3 copies of the HBV genome (ayw subtype) C57BL / 6 mice were injected intravenously and blood and liver tissue samples were collected at different time points. Serum HBsAg and HBeAg levels were detected by ELISA. Serum and liver viral load were detected by Real-time PCR. HE staining and immunohistochemical staining were used to detect liver histopathology Neutropenia and Neisseria gonorrhoeae were detected by real-time RT-PCR and RT-PCR. Neonatal mice were immunized with dexamethasone (DEX) and immunosuppressed mice were injected intraperitoneally with dexamethasone (DEX) Mouse model, and serum HBsAg, HBeAg detection. The results showed that in normal immune state, the serum and liver tissue of HBV-associated antigen were positive after 10 days transfection of pAAV-HBV1.3. After 30 days, the HBV-related antigen was negative, but the serum and liver tissue viral load were positive at 30d and 60d (P <0.01, P <0.05). After transfection with dexamethasone, the hepatitis B virus (HBsAg) established by high-pressure hydrodynamic method in immunosuppressed mice could still detect HBsAg , HBeAg expression. The above results show that acute hepatitis B mouse model is established by high pressure hydrodynamic method, which can prolong the retention time of virus in mice by inhibiting the immune status of mice. The model was established for the evaluation of HBV vaccine, drug development and pathogenesis of hepatitis B laid the foundation for the study.