永生化人肝细胞系的建立及其鉴定  

Establishment and further identification of immortalization human hepatocyte lines

作　　者：潘小平[1] 杜维波[1] 盛国平[1] 虞晓鹏[1] 喻成波[1] 吕国良[1] 曹红翠[1] 李君[1] 章益民[1] 陈瑜[1] 李兰娟[1] 

机构地区：[1]浙江大学医学院附属第一医院传染内科传染病诊治国家重点实验室,杭州310003

出　　处：《中华传染病杂志》2009年第7期394-397,共4页Chinese Journal of Infectious Diseases

基　　金：国家863计划资助项目（H40803）;国家自然科学基金重点项目（30630023）;十一五重大专项资助项目（2008ZX10002-005）;浙江省卫生厅医药卫生科研基金项目（2007A081）

摘　　要：目的建立永生化的人肝细胞系，为生物人工肝、肝细胞移植、体外药物代谢等提供细胞模型。方法应用胶原酶等四步灌流法分离获得原代人肝细胞，以含有SV40大T抗原（SV40LT）的重组反转录病毒对原代人肝细胞进行永生化，并对永生化人肝细胞进行生物学特性鉴定。结果原代人肝细胞经含有SV40LT的重组反转录病毒感染3～4周后，获得2株永生化人肝细胞系，分别命名为HepLi2和HepLi3，永生化人肝细胞在相差显微镜下具有典型的肝细胞形态。Western印迹显示有SV40LT基因的蛋白表达；RT-PCR显示永生化人肝细胞有Alb、谷胱甘肽S转移酶（GSTp）、人凝血因子X（HBCF-X）和β-actinmRNA表达，有细胞色素（CY）P450亚型（CYP3A5、CYP2E1、CYP2C8—19、CYP3A4）mRNA的表达；在HepLi2和HepLi3永生化人肝细胞的培养上清液中可检测到ALT、乳酸脱氢酶（LDH）和Alb。结论新建立的永生化人肝细胞系具有正常人肝细胞的生物学特性以及完善的肝细胞CYP450酶功能，可进一步用于肝细胞药物代谢研究。Objective To establish immortalized human hepatocyte lines for studies of bioartificial liver, hepatocyte transplantation, and drug metabolism in vitro. Methods Primary human hepatocytes were isolated by 4-step perfusion technique with collagenase and transfected with recombinant retrovirus containing Simian virus 40 large T antigen （SV40 LT）. Subsequently, immortalized human hepatoeytes were evaluated by analysis of gene expression and functional characteristics in vitro. Results Two immortalized human hepatocyte lines, HepLi2 and HepLi3, were obtained after primary human hepatocytes being infected by SV40 LT containing recombinant retrovirus for 3- 4 weeks. The immortalized human hepatocytes showed classical appearance of hepatoeyte observed by phase contrast microscope. The protein expression of SV40 LT in HepLi2 and HepLi3 cells were detected by Western blotting. The mRNA expressions of albumin （Alb）, glutathione S-transferase （GST-p）, human blood coagulation factor X （HBCF-X） and β-actin in HepLi2 and HepLi3 cells were detected by reverse transcription polymerase chain reaction （RT-PCR）, and the mRNA expressions of cytochrome （CY）450 subtypes （CYP3A5, CYP2E1, CYP2C8-19 and CYP3A4） in HepLi2 and HepLi3 ceils were also observed by RT PCR. Levels of alanine transaminase （ALT）, lactate dehydrogenase （LDH） and Alb were detected in the supernatant of immortalized human hepatocyte culture. Conclusions The immortalized human hepatocyte lines have the biological characteristics of primary human hepatocytes and have the CYP450 functions of hepatocytes, which may be helpful for the studies of bioartificial liver, hepatocyte transplantation and drug metabolism in vitro.

关 键 词：肝 人工 细胞系 猴病毒40 抗原 病毒 肿瘤 肝细胞 细胞色素P450酶系统 

分 类 号：R686[医药卫生—骨科学]