Expression of cytochrome P-450 enzymes in cultured human hepatocytes.
Expression of cytochrome P-450 enzymes in cultured human hepatocytes.
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DOI：
10.1111/j.1432-1033.1990.tb19140.x
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发表时间：
1990-07
期刊：
影响因子：
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通讯作者：
F. Morel;P. Beaune;D. Ratanasavanh;J. Flinois;C. Yang;F. Guengerich;A. Guillouzo
中科院分区：
文献类型：
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作者：
F. Morel;P. Beaune;D. Ratanasavanh;J. Flinois;C. Yang;F. Guengerich;A. Guillouzo
将成人和新生儿的肝细胞进行原代培养，并暴露于苯巴比妥、3-甲基胆蒽或利福平（三种众所周知的动物细胞色素P-450诱导剂）。研究了四种细胞色素P-450酶（或酶组，即P-450 IIIA、P-450 IIC 8/9/10、P-450 IIE 1和P-450 IA 2）的表达。发现这些酶在研究的培养期间保持表达。用诱导剂处理三天导致不同的反应，这取决于诱导剂和酶。苯巴比妥和利福平增加P-450 IIC 8/9/10 mRNA转录和相应的蛋白质，而3-甲基胆蒽无效。利福平对P-450 Ⅲ A mRNA和蛋白表达均有强烈的诱导作用。所有的肝细胞合成P-450 IIIA响应利福平，如所示的免疫过氧化物酶染色。P-450 IIIA的表达不受苯巴比妥的影响，并减少3-甲基胆蒽。在这些培养物中，P-450的IA 2和IIE 1下降至初始水平的25-50%。P-450 IA 2和乙氧基试卤灵O-脱乙基酶活性（其是与P-450 IA家族相关的单加氧酶活性）仅被3-甲基胆蒽增加，并且不响应于其它诱导剂。P-450 IIE 1不被这些化合物中的任何一种诱导。还在一名新生儿的人肝细胞中测定了P-450 IIC 8/9/10和P-450 IIIA mRNA水平。P-450 IIC 8/9/10在新鲜分离的细胞中几乎不表达，但随着培养时间的推移显着增加。P-450 IIIA转录本丰富的新鲜分离和培养的细胞来自新生儿。这些结果清楚地表明，人肝细胞继续表达细胞色素P-450酶，并对培养物中的诱导剂产生应答。该模型系统为研究药物对人肝脏药物代谢酶成熟和表达的影响提供了一种有用的方法。
Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3-methylcholanthrene, or rifampicin, three well-known inducers of cytochrome P-450 in animals. The expression of four cytochrome P-450 enzymes (or groups of enzymes, namely P-450 IIIA, P-450 IIC8/9/10, P-450 IIE1, and P-450 IA2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses, depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P-450 IIC8/9/10 mRNA transcripts and the corresponding protein, while 3-methylcholanthrene was ineffective. Both P-450 IIIA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P-450 IIIA in response to rifampicin, as shown by immunoperoxidase staining. P-450 IIIA expression was not affected by phenobarbital and was decreased by 3-methylcholanthrene. P-450s IA2 and IIE1 decreased to 25-50% of the initial level during these cultures. P-450 IA2 and ethoxyresorufin O-deethylase activity (which is a monooxygenase activity related to P-450 IA family) were increased only by 3-methylcholanthrene and did not respond to the other inducers. P-450 IIE1 was not induced by any of these compounds. P-450 IIC8/9/10 and P-450 IIIA mRNA levels were also measured in human hepatocytes from one newborn. P-450 IIC8/9/10 was barely expressed in freshly isolated cells but increased dramatically with time in culture. P-450 IIIA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn. These results clearly demonstrate that human hepatocytes continue to express cytochrome P-450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug-metabolizing enzymes in human liver.