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

原代培养大鼠肝细胞中激素因子对细胞色素 P-450 表达的调节。

基本信息

批准号：
02404025
负责人：
KATO Ryuichi
金额：
$ 17.92万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

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.

本研究采用原代培养的大鼠肝细胞色素P-450，在分子水平上研究了激素因素对大鼠肝细胞色素P-450表达的直接影响。各种肝细胞色素P-450的水平在肝细胞的原代培养过程中迅速下降。因此，我们用基质胶来防止P-450的减少，并成功地用苯巴比妥（PB）在肝细胞中诱导P-450 2B 1（CYP 2B 1）。甲状腺激素对PB的诱导有明显的抑制作用。此外，生长激素与甲状腺激素对PB诱导的抑制作用具有协同作用。另一方面，脱氢表雄酮拮抗生长激素的诱导。通过使用苯巴比妥的衍生物，我们显示了巴比妥酸盐对每个细胞色素P-450，如CYP 2B 1，CYP 2B 2和CYP 3A的诱导效力的结构特异性。采用Ca^++-共沉淀法成功地将P-450基因转染入细胞。我们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CYP 2B 1基因TATA盒附近。此外，我们还观察到CYP 3A 2基因6 β A中的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。目前正利用多种CYP 3A 2（6 β A）-CAT融合基因的表达载体研究地塞米松诱导的表达机制。