永生化人胎肝细胞系LC-1的建立

Establishment of immortalized human fetal hepatocyte line LC-1

目的建立人源性永生化肝细胞。方法利用含有SV40T和EGFP基因的双顺反子逆转录病毒表达载体pLNC-TIGlox,转染包装细胞PT67;取病毒上清液感染原代培养的人胎肝细胞,用G418筛选成功转导的永生化细胞系;用细胞计数法测定永生化细胞增殖特性,免疫细胞化学染色检测肝细胞特征蛋白表达,放免法和自动生化仪检测清蛋白和尿素氮合成功能。结果获得的一个细胞系命名为LC-1,其细胞群体倍增时间约为17h,可表达清蛋白、CK18肝细胞特征和CK19等胆管特征蛋白,具有清蛋白与尿素氮合成功能。结论成功建立了具有良好的增殖能力、分化状态、肝细胞生物学特征的永生化肝细胞系。

Objective To establish immortalized human hepatocyte lines. Methods PT67 cells were transfected with pLNCTI- Glox,an ambicistron retroviral expression vector previously constructed by our study group and containing ST40 and EGFP. Primary human fetal hepatocytes were infected with virus-contained supertanants and the successfully transducted cell lines were screened with G418. The proliferation characteristics of the cells were evaluated with cytometry. Hepatocyte-specific protein expression was detected with immunocytochemical staining. Synthesis of albumin and urea nitrogen was evaluated with radioimmunoassay and auto- biocheinical assay. Oncogenicity of the cell line was assessed with tumor-forming test. Results A cell line was obtained and named LC-1. The population doubling time of LC-1 ceils was 17h approximately. LC-1 cells could express hepatocyte-specific proteins such as albumin,cytokeratin （CK） 18 and CK19. LC-1 cells could synthesize albumin and urea. Conelosion We successfully established immortalized human bepatocytes retaining the characteristics of differentiated hepatocytes.