永生化胚胎肝细胞系构建及其生物学特性.docVIP

永生化胚胎肝细胞系构建及其生物学特性 　 作者：李羽，白雪帆，王军，徐哲，张岩 【关键词】 永生化；肝细胞；细胞系；生物学特性；SV40；脂质体 　　Establishment and biological characterization of an immortalized human fetal hepatocyte line 　　　【Abstract】 AIM: To establish an immortalized human fetal hepatocyte line and to study its biological characteristics and functions. METHODS: Human fetal hepatocytes were transfected with pcDNA3.1 recombined vector containing the Simian Virus 40 large T antigen. Characteristics of the immortalized fetal hepatocyte line were evaluated by morphologic and functional detection. RESULTS: One of the hepatocyte clones displaying highly differentiated in liver function and immortalized characteristics had been screened after 40 day selection of 700-300 mg/L G418. The immortalized hepatocyte line maintained the most morphologic characteristics of primary hepatocyte. The cell cloneforming rat was 31.2%. The immortalized human fetal hepatocytes had a normal karyotype and were not able to grow in soft agar culture. It had the ability of composing albumin and the immunohistochemical test showed that the SV40T gene had been integrated into the transformed cell genome. CONCLUSION: The immortalized human fetal hepatocyte line has the similar most morphologic characteristics and biological function to primary hepatocytes, and it would become ideal cell material on the study of bioartificial liver and hepatocyte transplantation. 　　【Keywords】 immortalized; hepatocytes; cell line; characterization; SV40; liposomes 　　【摘要】 目的: 构建永生化肝细胞系并对其生物学特性及某些功能进行研究. 方法： 利用SV40T基因和真核表达载体pcDNA3.1经脂质体转染至体外分离培养的人胚胎肝细胞，使其永生化，进一步鉴定其形态学特征和生物学功能. 结果： 经G418 700～300 mg/L筛选，40 d后获得一株阳性克隆. 形态学观察发现，该细胞具有原代培养肝细胞的大多数典型特征. 永生化胚胎肝细胞克隆形成率为31.2%，染色体核型分析表明细胞核型无明显异常，软琼脂集落形成试验表明细胞在软琼脂中不能生长，免疫组化证明SV40T基因已整合入细胞，而且该细胞具有合成白蛋白的功能. 结论： 新建胚胎肝细胞系具有与原代肝细胞类似的形态特征及生物学功能，可以成为生物人工肝及肝细胞移植研究中的理想细胞材料. 　　【关键词】 永生化；肝细胞；细胞系；生物学特性；SV40；脂质体 　　【中图号】 R512.6 　　0引言 　　原位肝移植可以有效治疗各种原因引起的肝功能衰竭，因而成为治疗肝功能衰竭的唯一有效手段. 但由于受到技术复杂、价格昂贵尤其是供肝短缺的困扰，使它的临床应用受到很大限制，难以及时有效地挽救患者的生命. 研究显示生物人工肝及肝细胞移