SV40LT抗原介导的人源性肝细胞系的构建

Establishment of a hepatocyte line from human with SV40LT antigen

目的 建立人源性肝细胞系 ,为生物人工肝和肝细胞移植等提供合适的细胞源。方法 利用SV4 0LTag基因和真核表达载体pcDNA3.1( - )经脂质体转染至来源于 2 5岁男性脑死亡者供肝的原代培养细胞 ,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G4 1870 0～30 0 μg/ml筛选 ,4 2d后获一抗G4 18肝细胞系。该细胞系呈单层贴壁、上皮细胞样形态生长 ,体外培养可无限传代 ,具有分泌白蛋白 (ALB)、丙氨酸氨基转移酶 (ALT)、天冬氨酸氨基转移酶 (AST)及乳酸脱氢酶 (LDH)的功能 ,超微结构观察发现 ,转染肝细胞具有原代培养肝细胞的大多数典型特征 ,如较大的细胞核、胞浆内含有丰富的糖原颗粒、大量的线粒体和粗面内质网等。经逆转录聚合酶链反应(PCR)和蛋白质印迹检测 ,转染肝细胞的ALBmRNA阳性和细胞色素P4 5 0 2E1阳性。

Objective To establish a hepatocyte line from human for bioartificial liver support system and hepatocyte transplantation. Methods Hepatocytes isolated from donor liver of a 25 year old brain death male were transfected with pcDNA3.1(-) recombined vector containing the genes encoding Simian Virus 40 large T antigen. Characteristics of the immortalized hepatocyte line were evaluated by morphologic and functional methods. Results One of the hepatocyte clones displayed highly differentiated liver function and immortalized characteristics has been screened after 42 day's selection of 700～300 μg/ml G418. The immortalized hepatocyte appeared epithelial and displayed morphologic characteristics of liver parenchymal cells. ALB, AST, ALT and LDH secreted from hepatocyte line were detected. Transmission electron microscope examination revealed that immortalized cells contained a round nucleus with nucleoli, moderate numbers of mitochondria, numerous rough endoplasmic reticulums, and some vacuoles. Adjacent cells were joined with junctional complexes. RT PCR and Western blot detection revealed that immortalized hepatocyte could express ALB mRNA and showed immunoreactive of cytochrome P450 2E1. Conclusions The new established hepatocyte line from human retained some specificbiologic functions and morphological cha racteristics of primary cultured hepatocytes.