Basic Study for Regenerative Medicine : Differentiate of Human Fetal Liver Cells and Application for Drug Pharmacokinetic Experiments

人胎儿肝（HFL）细胞培养始于6个正常人肝组织。采用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基溴化四氮唑法测定HFL细胞的增殖和活力，培养15天后，细胞增殖超过100倍。随着培养时间的延长，HFL细胞中白蛋白（ALB）、肝细胞生长因子4 α、肝细胞生长因子、CYP3A4、CYP3A5、CYP3A7 mRNA的表达水平逐渐升高，α-胎蛋白（AFP） mRNA的表达水平逐渐降低。而在HepG2细胞中，ALB和AFP mrna的表达水平没有变化，而CYP3A4、CYP3A5和CYP3A7 mrna的表达水平逐渐下降。在HepG2细胞中可以检测到CYP3As主要亚型CYP1A2、CYP2A6、CYP2B6、CYP2C （2C9和2C19）、CYP2D6和CYP2E1的mRNA表达。除CYP1A2外，HepG2细胞中表达的所有CYP mrna均在HFL细胞中检测到。在HFL细胞中，地塞米松（DEX）显著上调CYP3A、More 4和CYP3A7 mRNA的表达水平，而利福平（RIF）则无显著上调。DEX、RIF或苯巴比妥（PB）均未显著增加CYP3A5 mRNA水平。另一方面，经DEX、PB和RIF处理后，HepG2细胞中CYP3A4、CYP3A5和CYP3A7 mRNA的表达水平从2- 3倍增加到3倍。HepG2细胞有孕烷X受体mRNA表达，HFL细胞无。DEX诱导睾酮6β -羟化酶活性约为对照的2倍。然而，RIF的联合治疗并没有改变dex介导的CYP3A mRNA表达和睾酮6β-羟化酶活性。糖皮质激素受体（GR）拮抗剂RU486以剂量依赖性方式抑制dex介导的CYP3A mRNA诱导。在5 μM RU486下，dex介导的CYP3A4、CYP3A5和CYP3A7 mRNA表达几乎完全被抑制。这些结果提示，在人胎儿肝细胞中，PXR不参与dex介导的CYP3A4和CYP3A7的诱导，而直接由GR介导

Human fetal liver (HFL) cell culture was initiated from a pool of six normal human liver tissues. The proliferation and viability of HFL cells were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay, and the cells increased by more than 100-fold by culture for 15 days. The levels of expression of albumin (ALB), hepatocyte growth factor 4 α, hepatocyte growth factor, CYP3A4, CYP3A5, and CYP3A7 mRNAs in HFL cells increased with culture period, while that of α-fetoprotein (AFP) mRNA decreased gradually. In HepG2 cells, however, the expression levels of ALB and AFP mRNAs were not changed, and the levels of expression of CYP3A4, CYP3A5, and CYP3A7 mRNAs decreased gradually. The mRNA expression of major CYP isoforms including CYP3As, i.e., CYP1A2, CYP2A6, CYP2B6, CYP2C (2C9 and 2C19), CYP2D6, and CYP2E1, could be detected in HepG2 cells. With the exception of CYP1A2, all of the CYP mRNAs expressed in HepG2 cells were detected in HFL cells. In HFL cells, CYP3A … More 4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital (PB). On the other hand, CYP3A4, CYP3A5, and CYP3A7 mRNA expression levels in HepG2 cells were increased from 2- to 3-fold by treatment with DEX, PB, and RIF. Pregnane X receptor mRNA was expressed in HepG2 cells, but not HFL cells. Testosterone 6β -hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6β-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5 μM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR. Less