ESTABLISHMENT OF A RECOMBINANT HEPATIC CELL-LINE STABLY EXPRESSING ALCOHOL-DEHYDROGENASE
ESTABLISHMENT OF A RECOMBINANT HEPATIC CELL-LINE STABLY EXPRESSING ALCOHOL-DEHYDROGENASE
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DOI：
10.1006/abbi.1995.1400
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发表时间：
1995-08-20
影响因子：
3.9
通讯作者：
TUMA, DJ
中科院分区：
文献类型：
--
作者：
CLEMENS, DL;HALGARD, CM;TUMA, DJ
长时间培养的肝细胞失去了表达乙醇脱氢酶的能力，从而失去了有效氧化乙醇的能力。因此，很难在体外研究肝细胞慢性乙醇氧化的影响。为了避免这个问题，我们将外源性酒精脱氢酶基因的编码区插入肝细胞系。利用人肝癌细胞系Hep G2，构建了稳定表达乙醇脱氢酶的肝细胞系。这些重组细胞被称为HAD 73.1细胞，表达了新鲜分离的大鼠肝细胞中大约40%的乙醇脱氢酶活性。当直接测量这些细胞的乙醇代谢能力时，结果表明，这些细胞不仅能够以大约70%的新分离的大鼠肝细胞代谢乙醇，而且在培养基中检测到高达50 μ M的乙醛浓度。此外，在乙醇氧化过程中产生的乙醛水平被氰酰胺（一种乙醛氧化抑制剂）增加，而这些细胞代谢乙醇的能力被吡唑（一种醇脱氢酶抑制剂）抑制。这些结果表明，该体外系统将是一个有价值的工具，可以进行详细的生化研究，探索慢性乙醇氧化对肝脏的影响以及酒精诱导的肝细胞损伤机制。(C) 1995学术出版社，Inc。
Hepatocytes cultured for extended periods of time lose the ability to express alcohol dehydrogenase and thus, the ability to efficiently oxidize ethanol. Therefore, it has been difficult to investigate the effects of chronic ethanol oxidation by hepatocytes in vitro. To circumvent this problem, we have inserted the coding region of an exogenous alcohol dehydrogenase gene into an hepatic cell line. Using the human hepatocellular carcinoma cell line, Hep G2, we have constructed an hepatic cell line that stably expresses alcohol dehydrogenase. These recombinant cells, termed HAD 73.1 cells, express approximately 40% of the alcohol dehydrogenase activity of freshly isolated rat hepatocytes. When the ethanol metabolizing ability of these cells was directly measured, the results indicated that not only were these cells able to metabolize ethanol at ap proximately 70% of the rate of freshly isolated rat hepatocytes but acetaldehyde concentrations of up to 50 mu M were detected in the medium. Furthermore, the level of acetaldehyde produced during ethanol oxidation was augmented by cyanamide, an inhibitor of acetaldehyde oxidation, while the ability of these cells to metabolize ethanol was inhibited by pyrazole, an inhibitor of alcohol dehydrogenase. These results suggest that this in vitro system will be a valuable tool enabling detailed biochemical studies exploring the effects of chronic ethanol oxidation on the liver and the mechanisms of alcohol-induced hepatic cell injury. (C) 1995 Academic Press, Inc.