永生化猪肝细胞的建立及其鉴定

Immortalization of porcine hepatocyte lines by transfection with simian virus 40 large T antigen and human telomerase reverse transcriptase

目的建立永生化的猪肝细胞系，为生物人工肝、肝细胞移植、体外药物代谢模型提供合适的细胞源。方法应用胶原酶等四步灌流法分离获得原代猪肝细胞，以SV40大T抗原（SV40LT）和含人端粒酶反转录酶（hTERT）的重组反转录病毒感染，对其进行永生化；然后，对建立的猪肝细胞系进行生物学特性鉴定。结果原代猪肝细胞经转染含人端粒酶反转录酶和SV40大T抗原的重组反转录病毒后4～6周获得三个肝细胞样细胞克隆，其中，一个猪肝细胞克隆（HepLP）传代60代以上；永生化猪肝细胞在相差显微镜、扫描电镜和透射电镜下具有典型的肝细胞的形态；Western印迹法检测该细胞系表达人端粒酶反转录酶和SV40大T抗原。RT-PCR检测到永生化猪肝细胞表达白蛋白mRNA、ELISA双抗体夹心法检测到永生化猪肝细胞培养上清液中自蛋白的分泌，以5．0×10^6细胞数接种裸鼠，观察6个月无肿瘤形成。结论新建立的无致瘤性的永生化猪肝细胞系具有正常猪肝细胞的生物学基本功能。

Objective To establish normally immortalized porcine hepatocyte lines by ectopic expression of simian virus 40 large T （SV40LT） antigen and the human telomerase reverse transcriptase（hTERT）. Methods Primary porcine Hepatocyte cells were transfected with recombinant retrovirus containing SV40LT or hTERT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Immortalized porcine hepatocyte was confirmed by examination. Results The morphological phenotype of the transfected cells was similar to the primary porcine hepatocyte. One clone, HepLP, has been maintained in cultue for half year, and expanded by more than 60 passages. SV40 LT and hTERT could be detected in transfected porcine hepatocyte. Pig albumin mRNA was also detected by RT-PCR. No tumor formation occurred when HepLP cells were injected into Balb/c nude mice. Conclusions The immortalized, nontumorigenic, porcine hepatocytes maintained the properties of porcine primary hepatocytes such as the albumin secretion. This generation of immortalized porcine hepatocyte may be helpful for bioartifical liver support system, hepatocytes transplantation, drug/toxicological studies, and liver biologic studies.