Regulation of expression of cytochrome P-450 by hormonal factor in primary cultured rat hepatocytes.

摘要采用肝细胞原代培养，在分子水平上研究激素因子对大鼠肝细胞色素P-450表达的直接影响。在肝细胞原代培养过程中，多种肝细胞色素P-450的水平迅速下降。因此，我们使用matrigel来阻止P-450的减少，并成功地用苯巴比妥（PB）诱导肝细胞中P-450 2B1（CYP2B1）。甲状腺激素对PB诱导有明显抑制作用。此外，生长激素与甲状腺激素对PB诱导有协同抑制作用。另一方面，脱氢表雄酮可拮抗生长激素的诱导作用。通过使用苯巴比妥衍生物，我们展示了巴比妥类药物对CYP2B1、CYP2B2和CYP3A等细胞色素P-450的诱导效力的结构特异性。我们成功地用Ca^<++>-共受体法转染了P-450基因。以氯霉素乙酰转移酶基因为受体基因，探讨了苯巴比妥诱导的机制。我们发现了一个与PB诱导有关的位置，位于CYP2B1基因TATA盒附近。此外，我们在CYP3A2基因6betaA中发现了5个糖皮质激素反应元件（GRE）序列和2个与肝核因子4（HNF-4）相关的序列。目前正在利用不同长度的CYP3A2(6betaA)-CAT融合基因研究地塞米松的表达机制。

SummaryWe used primary culture of hepatocytes to investigate a direct effect of hormonal factors on the expression at the molecular level of rat hepatic cytochrome P-450. The levels of a variety of hepatic cytochrome P-450 are rapidly decreased during primary culture of hepatocytes. We used, therefore,matrigel to prevent the decrease of P-450s and we successed induction of P-450 2B1(CYP2B1)by phenobarbital(PB)in the hepatocytes. Thyroid hormone showed a marked inhibition of PB induction. In addition,growth hormone showed synergistic inhibition with thyroid hormone of PB induction. On the other hand,dehydroepiandrosterone antagonized the induction by growth hormone. By using derivatives of phenobarbital, we showed the structure specificity of barbiturates on the potency of induction of each cytochrome P-450,such as CYP2B1, CYP2B2 and CYP3A. We succeeded in an effective transfection of P-450 gene by Ca^<++>-coprecipition method. We used chloramphenicol-acetyltransferase gene as a receptor gene and the mechanism of induction with phenobarbital was investigated. We revealed one position,which is related with PB induction and locates near TATA box of CYP2B1 gene. Moreover,we observed five sequences of glucocorticoid responsive element(GRE)in CYP3A2 gene 6betaA and two sequences related with hepatic nuclear factor 4(HNF-4). The mechanism of expression of by dexamethasone is now under investigation by using a variety of lengthes of CYP3A2(6betaA)-CAT fusion gene.