CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver
CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver
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DOI：
10.1053/jhep.2003.50094
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发表时间：
2003-03-01
期刊：
影响因子：
13.5
通讯作者：
Suzuki, T
中科院分区：
文献类型：
--
作者：
Iwahori, T;Matsuura, T;Suzuki, T
CYP 3A负责肝脏中约50%的治疗药物代谢活性。本研究采用由功能性肝细胞癌（HCC）细胞株FLC-5组成的生物人工肝，建立CYP 3A 4诱导的人体药物代谢模型。径向流生物反应器（RFB）是一种载体填充型生物反应器，用于FLC-5细胞的三维灌注培养。利福平处理后48小时，RBF中的CYP 3A 4信使RNA（mRNA）表达水平约为单层培养物中的100倍。Western blot分析也表明CYP 3A蛋白表达增加。当将睾酮（CYP 3A 4的底物）加入利福平处理的细胞培养物中时，形成代谢产物6 β-羟基睾酮。以CYP 3A 4 ER 6为探针的电泳迁移率变动分析（EMSA）表明，与单层培养相比，利福平处理的FLC-5细胞在RFB培养中可能产生相对高分子量的PXR/RXR α复合物。类似地，用HNF-4 α结合基序的探针进行的测定表明在RFB培养物中形成了大的蛋白质复合物。由于已知PXR通过其反应元件反式激活CYP 3A 4基因，并且PXR的表达受HNF-4 α调节，因此RFB培养物中与PXR或HNF-4 α的反应元件结合的大复合物可能有助于CYP 3A 4 mRNA的上调。因此，由人肝癌细胞系组成的生物人工肝可用于研究诱导人CYP 3A 4过程中的药物相互作用。
CYP3A is responsible for approximately 50% of the therapeutic drug-metabolizing activity in the liver. The present study was undertaken to establish the CYP3A4 inducible model for analysis of human drug metabolism using a bioartificial liver composed of the functional hepatocellular carcinoma cell (HCC) fine FLC-5. A radial-flow bioreactor (RFB), which is a carrier-filled type bioreactor, was used for 3-dimensional perfusion culture of FLC-5 cells. The CYP3A4 messenger RNA (mRNA) expression level 48 hours after rifampicin treatment in the RBF was approximately 100 times higher than that in a monolayer culture. Western blot analysis also demonstrated an increase in expression of the CYP3A protein. When testosterone, a substrate for CYP3A4, was added to the rifampicin-treated cell culture, 6beta-hydroxy testosterone as a metabolite was formed. Electrophoretic mobility shift assay (EMSA) with a CYP3A4 ER6 probe demonstrated that relatively high molecular weight complex containing pregnane X receptor (PXR)/retinoid X receptoralpha(RXRalpha), compared with that in the monolayer culture, is possibly generated in the RFB culture of FLC-5 treated with rifampicin. Similarly, the assay with a probe of HNF-4alpha-binding motif indicated the formation of a large protein complex in the RFB culture. Because it is known that PXR transactivates CYP3A4 gene via its response element and expression of PXR is regulated by HNF-4alpha, the large complexes binding to response elements of PXR or HNF-4a in the RFB culture may contribute to up-regulation of CYP3A4 mRNA. In conclusion, the bioartificial liver composed of human functional HCC cell line was useful in studying drug interactions during induction of human CYP3A4.