Establishment and characterization of hepatocytes from an Immortomouse/SMP30/GNL knockout mouse hybrid lacking vitamin C to study vitamin C transport
Establishment and characterization of hepatocytes from an Immortomouse/SMP30/GNL knockout mouse hybrid lacking vitamin C to study vitamin C transport
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缺乏维生素 C 的 Immortomouse/SMP30/GNL 敲除小鼠杂交肝细胞的建立和表征，用于研究维生素 C 转运
DOI：
10.1093/jb/mvr109
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发表时间：
2011
影响因子：
2.7
通讯作者：
A.
中科院分区：
文献类型：
--
作者：
Amano;A.;Handa;S.;Aigaki;T.;Shigemoto;K.;Maruyama;N.;Ishigami;A.
衰老标志蛋白30（SMP 30）是一种内酯水解酶，参与维生素C（L-抗坏血酸，AA）的生物合成。我们先前报道了SMP 30/GNL基因敲除（KO）小鼠不能在体内合成AA的发展。为了更有效地研究肝脏的AA摄取和尚未表征的外排系统，我们建立了一个永生的肝细胞系，来自SMP 30/GNL KO小鼠和Immortomice的杂交。Immortomice表达不耐热猿猴病毒40（SV 40）大T抗原tsA 58。这些SMP 30/GNL KO永生肝细胞在33°C的允许温度下增殖，但在39°C的非允许温度下迅速降解。此外，它们是SMP 30/GNL缺陷型，表达SV 40大T抗原，并在33°C下稳定增殖。然而，细胞的增殖在39°C下被阻止。相差显微照片显示，细胞是双核的，具有与野生型小鼠的原代培养肝细胞相似的增大的细胞质。AA摄取的剂量反应和时间依赖性研究表明，细胞，虽然不能合成AA，采取了AA从培养基中。我们的SMP 30/GNL永生肝细胞的这一特性使其对于研究肝脏中的AA摄取和外排系统非常有用。
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 AAin 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.