Stable overexpression of arginase I and ornithine transcarbamylase in HepG2 cells improves its ammonia detoxification
Stable overexpression of arginase I and ornithine transcarbamylase in HepG2 cells improves its ammonia detoxification
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DOI：
10.1002/jcb.23375
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发表时间：
2012-02-01
影响因子：
4
通讯作者：
Chen, Yanlin
中科院分区：
文献类型：
--
作者：
Tang, Nanhong;Wang, Yan;Chen, Yanlin
HepG 2是一种永生化的人肝癌细胞系，已用于生物人工肝系统的研究。然而，氨解毒水平低是其最大的缺点。本研究利用真核双基因表达载体pBudCE4.1构建了稳定表达人精氨酸酶I（hArgI）和人鸟氨酸氨甲酰转移酶（hOTC）的重组HepG 2细胞系HepG 2/（hArgI+hOTC）4。(1)HepG 2/（hArgI+hOTC）4细胞的hArgI和hOTC酶活性均高于对照细胞。(2)HepG 2/（hArgI+hOTC）4细胞的耐氨能力是HepG 2细胞的3倍，是原代培养人肝细胞的37.5%。在氨解毒实验中，HepG 2/（hArgI+hOTC）4细胞产生的尿素是对照组的3.1倍（在180？mM NH 4Cl）和3.1倍的谷氨酰胺（在120 mM NH 4Cl和15？mM谷氨酸）比HepG 2细胞，分别达到原代人肝细胞的63.1%和36.0%。(3)hArgI和hOTC过表达对HepG 2细胞的生长无影响，但可促进谷氨酰胺合成酶（GS）、精氨酸酶II（ArgII）、琥珀酸合成酶（ASS）和琥珀酸裂解酶（ASL）等氨解毒相关蛋白的表达。本研究表明，该修饰方法在改善HepG 2细胞功能方面取得了显著进展，HepG 2/（hArgI+hOTC）4细胞将为生物人工肝系统的应用提供更好的选择。J.细胞。113：518527，2012. (C)2011 Wiley Periodicals，Inc.
HepG2 is an immortalized human hepatoma cell line that has been used for research into bioartificial liver systems. However, a low level of ammonia detoxification is its biggest drawback. In this work, a recombinant HepG2 cell line with stable overexpression of human arginase I (hArgI) and human ornithine transcarbamylase (hOTC), HepG2/(hArgI+hOTC)4, was developed using a eukaryotic dual gene expression vector pBudCE4.1. (1) The hArgI and hOTC enzymatic activity in HepG2/(hArgI+hOTC)4 cells were higher than in the control cells. (2) The ammonia tolerance capacity of HepG2/(hArgI+hOTC)4 cells was three times that of HepG2 cells and 37.5% of that of primary human hepatocytes in cultivation. In the experiment of ammonia detoxification, HepG2/(hArgI+hOTC)4 cells produced 3.1 times more urea (at 180?mM NH4Cl) and 3.1 times more glutamine (at 120 mM NH4Cl and 15?mM glutamate) than HepG2 cells, reaching 63.1% and 36.0% that of primary human hepatocytes, respectively. (3) The hArgI and hOTC overexpression did not influence the growth of HepG2 cells and also promoted the expression of other ammonia detoxification associated proteins including glutamine synthetase (GS), arginase II (ArgII), arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL) in HepG2 cells. This work illustrates that the modification reported here made significant progress in the improvement of HepG2 cell function and the HepG2/(hArgI+hOTC)4 cells will provide a better selection for the application of bioartificial liver system. J. Cell. Biochem. 113: 518527, 2012. (C) 2011 Wiley Periodicals, Inc.