中国流行株C基因型HBV稳定复制表达小鼠模型的建立  

Establishment of a mouse model stably replicating and expressing hepatitis B virus genotype C prevailed in China

作　　者：杨悦[1,2] 高俪 许智慧[2] 余双庆 李瑞生[2] 黄鹏宇[2] 乔艳[1,2] 李进[2] 董小岩 吴小兵 刘妍[2] 徐东平[2] 

机构地区：[1]桂林医学院研究生院,广西桂林541004 [2]解放军302医院全军传染病研究所/临床研究管理中心,北京100039 [3]五加和分子医学研究所有限公司,北京100176

出　　处：《解放军医学杂志》2016年第10期793-797,共5页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金面上项目(81371852,81573676,81373136)~~

摘　　要：目的构建稳定复制中国流行株C基因型HBV的小鼠模型。方法将携带1.3倍C基因型HBV基因组（adr血清型）的重组腺相关病毒r AAV8-1.3HBV-C转导人肝癌细胞Hu H7,采用ELISA法评估HBV抗原（HBs Ag、HBe Ag）在肝癌细胞中的表达。筛选高表达的重组病毒,经尾静脉注射入6~8周龄C57BL/6小鼠体内,建立HBV-C复制小鼠模型（实验组,n=8）;同时建立文献已报道HBV-D复制小鼠模型（对照组,n=7,r AAV8-1.3HBV-D,ayw血清型）。动态监测两组小鼠血清（眼底静脉丛采血）HBV DNA载量和HBs Ag、HBe Ag的抗原表达量;于第9周处死小鼠,HE染色观察肝组织病理改变,免疫组化染色分析HBs Ag和HBc Ag的表达。结果重组病毒r AAV8-1.3HBV-C体外转导人肝癌细胞Hu H7,72h后细胞上清中可检测到HBs Ag和HBe Ag的表达。小鼠注射重组病毒后第2、3、5、7、9周,血清HBV DNA存在稳定复制,血清HBe Ag表达水平较稳定,但血清HBs Ag表达存在波动。两组小鼠肝组织未见明显的炎性细胞浸润及组织结构异常,但可检测到HBs Ag和HBc Ag蛋白。结论利用高嗜肝性重组8型腺相关病毒载体携带1.3倍C基因型HBV基因组体内转导C57BL/6小鼠,成功地建立了稳定复制并持续表达C基因型HBV的小鼠模型。Objective To establish a mouse model stably replicating and expressing hepatitis B virus genotype C（HBV-C） prevailed in China. Methods The recombinant adeno-associated virus r AAV8-1.3HBV-C（adr serotype） was transduced into the human hepatocellular carcinoma Huh7 cells in vitro, and the expression of HBs Ag and HBe Ag in the cell culture supernatant was determined by ELISA. High expression recombinant virus r AAV8-1.3HBV-C was screened and injected via the tail vein into eight C57BL/6 mice（aged 6-8 weeks） as the experimental group; meanwhile the previously reported r AAV8-1.3HBV-D（ayw serotype） was injected into seven C57BL/6 mice as the control group. HBV DNA load, HBs Ag and HBe Ag levels in sera were assayed at weeks 2, 3, 5, 7 and 9 post viral injections. Mice were sacrificed 9 weeks post injection and Hematoxylin-eosin（HE） staining and immunohistochemistry were performed to evaluate the pathological changes, and HBs Ag and HBc Ag expressions in the liver tissue. Results The supernatant HBs Ag and HBe Ag were detectable in the Hu H7 cells 72 h after transduction in vitro. The fluorescence quantitative PCR results of the HBV DNA load in serum at week 2, 3, 5, 7, and 9 post viral injection suggested stable in vivoreplication of HBV DNA in mice. The serum expression of HBe Ag was stable while the serum expression of HBs Ag fluctuated. No obvious inflammatory cell infiltration or abnormal structure of liver tissue was observed, while HBs Ag and HBc Ag expression in the liver tissue were detected for both groups. Conclusion By in vivo transduction with the recombinant virus r AAV8-1.3HBV-C, a mouse model that stably expressed and replicated HBV-C has been successfully established.

关 键 词：乙型肝炎病毒 基因型 模型 动物 

分 类 号：R735[医药卫生—肿瘤]