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.

摘要我们使用原代肝细胞培养物来研究激素因素对大鼠肝细胞色素 P-450 分子水平表达的直接影响。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，我们使用基质胶来防止P-450s的减少，并成功地通过苯巴比妥（PB）在肝细胞中诱导P-450 2B1（CYP2B1）。甲状腺激素对 PB 诱导表现出明显的抑制作用。此外，生长激素与甲状腺激素对PB诱导具有协同抑制作用。另一方面，脱氢表雄酮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各种细胞色素P-450（如CYP2B1、CYP2B2和CYP3A）诱导效力的结构特异性。我们通过Ca^++-共沉淀法成功地有效转染了P-450基因。我们使用氯霉素乙酰转移酶基因作为受体基因，研究了苯巴比妥的诱导机制。我们发现了一个与PB诱导相关的位置，位于CYP2B1基因的TATA框附近。此外,我们在CYP3A2基因6betaA中观察到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。目前正在通过使用多种长度的CYP3A2(6betaA)-CAT融合基因来研究地塞米松的表达机制。