Runge D, Köhler C, Kostrubsky V E, Jäger D, Lehmann T, Runge D M, May U, Stolz D B, Strom S C, Fleig W E, Michalopoulos G K
Klinik für Innere Medizin I, Martin Luther Universität Halle-Wittenberg, Halle, 06097, Federal Republic of Germany.
Biochem Biophys Res Commun. 2000 Jun 24;273(1):333-41. doi: 10.1006/bbrc.2000.2902.
Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.
将无血清培养长达6周的人肝细胞用于研究参与药物代谢的酶和膜转运蛋白的表达及诱导情况。通过蛋白质免疫印迹分析，并在适当情况下通过乙氧异吩唑酮 - O - 脱乙基酶（EROD）活性和睾酮 - 6β - 羟化酶（T6H）活性的酶促测定，检测I相药物代谢酶细胞色素P450（CYP）1A1、CYP1A2、CYP2C9、CYP2C19、CYP2E1和CYP3A4。通过逆转录 - 聚合酶链反应（RT - PCR）和蛋白质免疫印迹分析检测到，在培养过程中膜转运蛋白多药耐药蛋白（P - 糖蛋白，MDR - 1）、多药耐药相关蛋白（MRP - 1）和肺耐药蛋白（LRP）的表达得以维持。利福平、苯巴比妥或3 - 甲基胆蒽以及β - 萘黄酮等模型诱导剂能够诱导CYP1A或CYP3A4以及EROD或T6H活性长达30天。CYP2C9、CYP2C19和CYP2E1的表达在48天内得以维持但不可诱导。此外，利福平和苯巴比妥不能显著提高MDR - 1和MRP - 1蛋白水平。
