原代培养人肝细胞体外乙型肝炎病毒感染和乙型肝炎病毒表面抗原黏附模型初探

目的研究建立人肝细胞体外乙型肝炎病毒（HBV）感染模型和乙型肝炎表面抗原（HBsAg）黏附模型，为后期研究小分子抗体Fab的作用打下基础。方法体外原代培养人肝细胞，对人原代肝细胞进行HBsAg黏附实验及HBV感染实验。采用正常人胎肝细胞系（L-02）及鼠原代肝细胞进行实验对照。倒置相差显微镜下观察细胞形态，生化法检测其白蛋白分泌功能。免疫组化法检测HBsAg黏附情况和HBV感染肝细胞内HBsAg的表达。酶联免疫吸附试验（ELISA）检测肝细胞感染HBV后HBsAg的持续表达。PCR检测肝细胞内HBV复制中间体-闭合环状双链DNA（cccDNA）。结果人原代肝细胞体外培养达2周以上。HBsAg黏附实验检测到HBsAg可黏附于人原代肝细胞、L-02、鼠原代肝细胞胞膜。HBV体外仅感染人原代肝细胞，从第2天起即可检测到细胞培养上清中存在HBsAg，并持续到观察结束第16天，免疫组化检测人原代肝细胞内HBsAg呈阳性；PCR检测到人原代肝细胞内存在HBV—cccDNA，而L—02及鼠原代肝细胞实验检测结果均为阴性。结论人原代肝细胞体外分离培养成功，HBsAg黏附和HBV感染模型初步建立于人原代肝细胞。

Model of Primary Human Hepatocytes Absorption with HBsAg and Infection with HBV

Objective To establish the model of primary human hepatocytes absorption with hepatitis B antigen(HBsAg) and infection with hepatitis B virus(HBV) in vitro,for the future study on recombinant micromolecule antibody-Fab effect.Methods Human primary hepatocytes were isolated and cultured,then absorbed with HBsAg and infected with HBV positive serum in vitro.Human fetal liver cells line(L-02) and primary rat hepatocytes were tested under the same treatment.Cell appearances were observed under inversion fluorescent microscope. Albumin excretion was detected by biochemistry detection.ELISA and immunohistochemistry were used for detecting HBsAg in cells and the supernatant.HBV-cccDNA in cell nuclear was detected by PCR.Results Human primary hepatocytes were cultured more than two weeks.HBsAg could be detected after HBsAg-absorption and HBV-infection in the hepatocytes.Secretions of HBsAg reached submit from day 2 after HBV-infection in the supernatant.HBsAg could be detached in those cells.HBV-cccDNA existed in the infected primary hepatocytes.Conclusion Primary human hepatocytes are competent for absorption with HBsAg and infection with HBV.