Long-term controlled immortalization of a primate hepatic progenitor cell line after Simian virus 40 T-Antigen gene transfer
Long-term controlled immortalization of a primate hepatic progenitor cell line after Simian virus 40 T-Antigen gene transfer
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DOI：
10.1038/sj.onc.1208089
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发表时间：
2005-01-20
期刊：
影响因子：
8
通讯作者：
Weber, A
中科院分区：
文献类型：
--
作者：
Delgado, JP;Parouchev, A;Weber, A
成肝细胞是肝细胞和胆管细胞的双能祖细胞。由于缺乏稳定的体外培养系统，这类细胞需要通过永生化的方式产生肝祖细胞系。在这项研究中，我们描述了长期的行为克隆猴胎肝祖细胞永生化的猿猴病毒40（SV 40）大T抗原（T-Ag）侧翼loxP网站。永生化与端粒酶活性的再表达有关，端粒酶活性在培养一年多后的晚期传代（群体倍增120）中降低。这种减少是伴随着端粒缩短和核型不稳定。然而，携带p53基因的染色体保持完整，长期永生化祖细胞保持接触抑制和增殖特性。它们还显示出正常双能表型的特征。我们构建了表达诱导型Cre重组酶的逆转录病毒载体，并将其转移到永生化祖细胞中。通过4-羟基-他莫昔芬激活Cre重组酶诱导SV 40 T-Ag切除，导致表达Cre重组酶的细胞死亡。永生化的祖细胞在晚期传代时停止生长，并在移植到免疫功能低下小鼠的肝脏后最终消失。这些细胞为研究肝分化和癌变提供了一种新的模型。
Hepatoblasts are bipotent progenitors of both hepatocytes and cholangiocytes. The lack of stable in vitro culture systems for such cells makes it necessary to generate liver progenitor cell lines by means of immortalization. In this study, we describe the long-term behaviour of a clone of simian foetal hepatic progenitor cells immortalized by Simian virus 40 (SV40) large T-antigen (T-Ag) flanked by loxP sites. Immortalization was associated with the reexpression of telomerase activity, which decreased at late passages ( population doubling 120) after more than a year in culture. This decrease was concomitant to telomere shortening and karyotypic instability. However, the chromosomes carrying the p53 gene remained intact and long-term immortalized progenitor cells maintained contact inhibition and proliferative properties. They also displayed the features of a normal bipotent phenotype. We constructed a retroviral vector expressing an inducible Cre recombinase and transferred it into the immortalized progenitors. Activation of the Cre recombinase by 4-hydroxy-tamoxifen induced SV40 T-Ag excision, leading to the death of cells expressing Cre recombinase. Immortalized progenitors at late passages stopped growing and eventually disappeared after transplantation into the livers of immunocompromised mice. These cells provide a novel model to study hepatic differentiation and carcinogenesis.