Key Laboratory of Organ Transplantation, Ministry of Education, Ministry of Health, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
World J Gastroenterol. 2010 Apr 7;16(13):1660-4. doi: 10.3748/wjg.v16.i13.1660.
To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40Tag).
We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination.
The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes.
In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.
为了建立一个肝细胞系，我们用含有猿猴病毒 40 大 T 抗原（SV40Tag）的逆转录病毒载体 SSR#69 对原代猪肝细胞进行永生化。
我们首先用改良的四步逆行灌注技术建立了一种猪肝细胞分离方法。然后，用携带 SV40T 和潮霉素抗性基因的逆转录病毒载体 SSR#69 对猪肝细胞进行永生化，这些基因被两侧带有 loxP 重组靶点的配对loxP 重组靶点所包围。在扩展的细胞中，SV40T cDNA 随后通过 Cre/LoxP 位点特异性重组进行切除。
用逆转录病毒载体 SSR#69 成功地永生化了具有高活力（97%）的原代猪肝细胞。其中一个永生化克隆表现出典型的形态学外观，即 TJPH-1，并通过克隆环选择和培养进行了扩增。用 Cre 重组酶切除 SV40T 基因后，细胞停止生长。回复细胞群体表现出分化肝细胞的特征。
总之，我们在此描述了一种改良的肝细胞分离方法，随后建立了一种通过逆转录病毒转导和位点特异性重组介导的猪肝细胞系。
