HBV适应性杂交细胞株的建立和初步研究

目的方法诱导HepG₂细胞产生HGPRT基因突变,将筛选出的HGPRT阴性的HepG2细胞与携带HBV的人原代肝细胞进行融合得杂交细胞,用HAT培养基筛选出异核体杂交细胞,再利用有限稀释法进行克隆,通过核型分析方法鉴定所得细胞。对所得杂交细胞及培养上清液进行HBV DNA和HBsAg、HBeAg的检测。实验同时,用未杂交的HepG2和人原代肝细胞作对照。结果经筛选后,有一杂交细胞株(HepCHLine3)克隆成功。HepCHLine3在形态学上与HGPRT^-HepG₂细胞相似,能体外传代培养,染色体核型分析示HepCHLine3杂交细胞染色体众数为99条,证明为融合细胞株,含所有来自HepG₂和人原代肝细胞的基因数。传代培养的HepCHLine3和其培养上清液用巢式PCR分别可检测到HBV DNA,培养上清液内检测到HBsAg和HBeAg。对照组的HepG₂和人原代肝细胞相应结果为阴性。提示该细胞携带并分泌HBV DNA。结论该杂交细胞兼具HepG₂细胞体外传代和人原代肝细胞对HBV易感的特性,是一新型杂交细胞系,为进一步建立新型HBV感染细胞模型奠定了基础。

Establishing the hybrid cell line for HBV

Objective To establish a hybrid cell different from either HepG2 cells or human primary hepatic cells which carry HBV and can be serial sub-cultured in vitro.Methods HGPRT-HepG2 cells,which were induced by 6-MP,were fused with human primary hepatic cells infected by the hepatitis B virus.After being screened with HAT,the hybrid cells were cloned through limiting dilution assay.Subsequently,the hybrid cells were identified by karyotype analysis.HBV DNA and HBsAg and HBeAg were determined at different generations.HepG2 and human primary hepatic cells infected by the hepatitis B virus were used as controls.Results Hybrid cells were morphologically similar to HGPRT-HepG2cells and can be sub-cultured in vitro.The karyotype analysis showed that the modal chromosome number was 99 in the hybrid cells,indicating that the hybrid cells contained all genomic factors from both HepG2 and human hepatic cells.HBV DNA can be detected in the cells and supernated by nest PCR.In the supernate, HBsAg and HBeAg can be detected.Conceivably the hybrid cells possessed the replication and generation in vitro of HepG2 as well as the sensitivity of human primary hepatic cells to HBV,which paved secretion of HBV DNA.Conclusion As a new hybrid cell line,it has the characteristics of both types for further study of the HBV infection cell model.