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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 construct a mouse model of stable replication of HBV genotype C in China. Methods Recombinant adeno-associated virus rAAV8-1.3HBV-C carrying 1.3-fold C genotype HBV genome (adr serotype) was transduced into human hepatocellular carcinoma cell line Hu H7 and the expression of HBsAg and HBe Ag in hepatocellular carcinoma Expression in cells. Highly expressed recombinant virus was screened and injected into tail vein of 6 to 8 weeks old C57BL / 6 mice to establish a HBV-C replicating mouse model (experimental group, n = 8). At the same time, the established literature has reported that HBV-D replication Mouse model (control, n = 7, rAAV8-1.3HBV-D, ayw serotype). HBs Ag and HBeAg were detected dynamically in the serum of the two groups of mice (blood collected from the fundus venous plexus). At the 9th week, the mice were sacrificed and the pathological changes of the liver were observed by HE staining. The expression of HBs Ag and HBc Ag expression. Results The expression of HBs Ag and HBe Ag was detected in the cell supernatant of recombinant adenovirus AAV8-1.3HBV-C transfected human hepatoma cell line Hu H7 at 72 h. At the 2nd, 3rd, 5th, 7th and 9th weeks after injection of the recombinant virus, the stable replication of serum HBV DNA and the stable expression of serum HBe Ag were observed. However, the expression of HBsAg in serum fluctuated. No significant inflammatory cell infiltration and histological abnormalities were observed in the liver of the two groups, but HBsAg and HBcAg proteins were detected. Conclusion The C57BL / 6 mice transduced with the HBV genome of 1.3 times C genotype were highly transgeneic using the recombinant adenovirus-associated adenovirus vector of type 8, and a mouse model of stable replication and continuous expression of HBV genotype C was established successfully.