Cellular Genetics, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, Japan.
J Biochem. 2011 Dec;150(6):671-8. doi: 10.1093/jb/mvr109. Epub 2011 Sep 7.
Senescence marker protein-30 (SMP30) has been identified as the lactone-hydrolysing enzyme gluconolactonase (GNL), which is involved in vitamin C (L-ascorbic acid, AA) biosynthesis. We previously reported the development of SMP30/GNL knockout (KO) mice unable to synthesize AA in vivo. For more efficient study of the liver's AA uptake and as yet uncharacterized efflux system, we established an immortal hepatocyte line derived from a hybrid of SMP30/GNL KO mice and Immortomice. Immortomice express the thermolabile simian virus 40 (SV40) large T antigen tsA58. These SMP30/GNL KO immortal hepatocytes proliferate at the permissive temperature of 33°C but degrade rapidly at the non-permissive temperature of 39°C. Additionally, they are SMP30-/GNL-deficient, express SV40 large T antigen and proliferate steadily at 33°C. However, the cells' proliferation is arrested at 39°C. A phase contrast micrograph revealed that the cells are binucleated with an enlarged cytoplasm similar to that of primary cultured hepatocytes from wild-type mice. Dose-response and time-dependent study of AA uptake revealed that the cells, although unable to synthesize AA, took up AA from the culture medium. This property of our SMP30/GNL immortal hepatocytes makes them extremely useful for studying AA uptake and efflux systems in the liver.
衰老标志物蛋白-30（SMP30）已被鉴定为内酯水解酶葡萄糖酸内酯酶（GNL），它参与维生素 C（L-抗坏血酸，AA）的生物合成。我们之前报道了体内无法合成 AA 的 SMP30/GNL 敲除（KO）小鼠的开发。为了更有效地研究肝脏的 AA 摄取和尚未表征的外排系统，我们建立了一种源自 SMP30/GNL KO 小鼠和 Immortomice 杂交的永生化肝细胞系。Immortomice 表达热不稳定的猿猴病毒 40（SV40）大 T 抗原 tsA58。这些 SMP30/GNL KO 永生化肝细胞在允许温度 33°C 下增殖，但在非允许温度 39°C 下迅速降解。此外，它们缺乏 SMP30/GNL，表达 SV40 大 T 抗原，并在 33°C 下稳定增殖。然而，细胞的增殖在 39°C 时被阻止。相差显微镜观察显示，细胞双核，细胞质增大，类似于野生型小鼠原代培养肝细胞。AA 摄取的剂量反应和时间依赖性研究表明，尽管这些细胞无法合成 AA，但它们可以从培养基中摄取 AA。我们的 SMP30/GNL 永生化肝细胞的这一特性使它们在研究肝脏中 AA 的摄取和外排系统方面非常有用。
