腺相关病毒-乙型肝炎病毒小鼠模型的建立  

Establishment of adeno-associated virus-hepatitis B virus mouse model

作　　者：应若素[1] 牛易[1] 林路平[1] 朱莹[1] 杨可立[1] 谢志伟[1] 李华鹏 伍倩琪[1] 高洪波[1] 关玉娟[1] Ying Ruosu;Niu Yi;Lin Luping;Zhu Ying;Yang Keli;Xie Zhiwei;Li Huapeng;Wu Qianqi;Gao Hongbo;Guan Yujuan(Department of Hepatology,Guangzhou Eighth People＇s Hospital,Guangzhou,Guangdong 510060,China)

机构地区：[1]广州市第八人民医院肝病科,510060 [2]广州派真生物技术有限公司

出　　处：《中华生物医学工程杂志》2017年第4期279-282,共4页Chinese Journal of Biomedical Engineering

基　　金：广东省医学科研基金（A2017053）

摘　　要：目的 建立一种有效的HBV感染小鼠模型.方法 采用三质粒共转染HEK293细胞生产腺相关病毒-乙型肝炎病毒（AAV-HBV）病毒,反复冻融后密度梯度离心法纯化病毒并进行鉴定.健康成年C57BL/B6小鼠20只按随机数字法分为实验组（n=10）和对照组（n=10）,实验组小鼠尾静脉注射AAV-HBV病毒,对照组小鼠注射同等剂量的AAV-GFP病毒.观察两组小鼠一般情况,不同时间点采血检测血清丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）水平,采用荧光定量PCR法、化学发光法分别进行血清HBV DNA和HBsAg定量检测.处死小鼠后获取肝脏进行病理检查,免疫组织化学法检测肝脏组织HBsAg、HBcAg表达.结果 成功获得纯化的AAV-HBV病毒,病毒纯度大于97％.AAV-HBV病毒感染小鼠一般情况良好,无异常表现,且肝功能正常.光镜下观察肝脏组织未见明显病理损伤表现,免疫组织化学检测显示肝细胞质可见HBsAg阳性表达、细胞核可见HBcAg阳性表达.AAV-HBV病毒感染小鼠血液中可稳定检测到HBV DNA和HBsAg表达.结论 成功构建了一种接近人类的,可有效、持续产生病毒的HBV感染小鼠模型.Objective To establish an effective hepatitis B virus （HBV）-infected mouse model.Methods HEK293 cells were co-transfected with the three plasmids to produce adeno-associated virus （AAV）-HBV virus.After repeated freeze-thaw cycles,the virus was purified by density gradient centrifugation and identified.Twenty healthy adult C57BL/B6 mice were randomly divided into the study group （n=10） and the control group （n=10）.The study group was injected with AAV-HBV virus through caudal vein,and the control group was injected with the same dose of AAV-GFP virus.The general condition in the two groups was determinted.The serum levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were determined at different time points.The quantitative detection of serum HBV DNA and HBsAg were examined by fluorescence quantitative PCR and chemiluminescence,respectively.After the mice were sacrificed,the livers were obtained for pathological examination.The HBsAg and HBcAg expression in the liver tissues were examined by immunohistochemistry.Results The purified AAV-HBV virus was successfully obtained with a purity of more than 97%.The general condition of mice infected with AAV-HBV virus was good with no abnormal behavior and normal liver function.No obvious pathological damage was observed in the liver tissues under the light microscope.The immunohistochemical staining showed positive HBsAg expression in the liver cytoplasm and positive HBcAg expression in the cell nucleus.The HBV DNA and HBsAg expression could be detected stably in the blood of mice infected with AAV-HBV virus.Conclusion The mouse model of HBV infection that is close to human beings has been established successfully,which can effectively and continuously produce virus.

关 键 词：腺相关病毒 乙型肝炎病毒 疾病模型 动物 小鼠 

分 类 号：R737[医药卫生—肿瘤]