表达HBV的SMMC-7721人肝癌细胞系模型的建立

Establishment of human hepatoma model expressing HBV genome

目的：建立表达ＨＢＶ的体外培养的细胞克隆，为研究ＨＢＶ的生物学性状提供良好的细胞模型。方法：将３．２ｋｂＨＢＶＤＮＡ插入携带新霉素抗药基因的真核细胞表达载体ｐＣＮＣＸ，应用磷酸钙沉淀技术将其导入体外培养的ＳＭＭＣ－７７２１人肝癌细胞系。结果：成功地获得表达ＨＢｓＡｇ和ＨＢｅＡｇ的细胞克隆。结论：应用磷酸钙沉淀技术将ＨＢＶＤＮＡ导入体外培养的细胞克隆，获得能表达ＨＢＶ的细胞克隆。

Objective: To establish the human hepatoma model expressing HBV genome. Methods: A 3.2 kb HBV DNA fragment was inserted into EcoRⅠ site of HBVpCNCX plasmid carrying gene of neomycin resistance. Human hepatoma 7721 cell were cultured in DMEM medium. Transfection of HBVpCNCX plasmid (30 μg) was mediated by exposure of the cells to calcium phosphate. The transfected cells were incubated in DMEM supplemented with 10% fetal bovine serum and G418(400 mg/L)for 30 d. Clones of cells that grew in the presence of G418 were isolated 30 d after transfection. HBsAg and HBeAg were identified with enzymelinked immunosorbent assay (ELISA). The mRNA for HBV genome was measured by dot blot and  in situ  hybridization. The integrated and extrachromosomal HBV DNA were identified by Southern blot hybridization. Results: Two transfected cell clones expressing HBsAg and HBeAg were obtained. Intracellular, integrated and extrachromosome HBV DNA sequences and RNA of HBV were detected. Conclusion: The cultured hepatoma cells can express HBV genome.