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

人胎肝(HFL)细胞培养是从6个正常人肝组织池中开始的。用3-[4，5-二甲基噻唑-2-基]-2，5-二苯基四氮唑溴化法检测HFL细胞的增殖和活力，培养15d后HFL细胞的增殖倍数增加100倍以上。随着培养时间的延长，白蛋白、肝细胞生长因子4α、肝细胞生长因子、细胞色素P3A4、细胞色素P3A5和细胞色素P3A7mRNAs的表达水平逐渐升高，而甲胎蛋白(α)的表达水平逐渐降低。而在HepG2细胞中，ALB和AFP的mRNAs表达水平无明显变化，而CYP3A4、CYP3A5和CYP3A7mRNAs的表达水平逐渐下降。在HepG2细胞中可检测到主要的细胞色素P3A亚型，即细胞色素P4501A2、细胞色素P450 2 A6、细胞色素P2 2 B6、细胞色素P2 2 C(2C9和2 C19)、细胞色素P2 D6和细胞色素P2 2 E1mRNA的表达。除CYP1A2外，所有在HepG2细胞中表达的CYP基因在HFL细胞中均可检测到。在HFL细胞中，细胞色素P3A…地塞米松(DEX)可显著上调MER4和CYP3A7mRNA的表达，而利福平(RIF)则不能上调MER4和CYP3A7的表达。地塞米松、RIF或苯巴比妥(PB)对细胞色素P3A5基因表达无明显影响。另一方面，经地塞米松、PB和RIF处理后，HepG2细胞中的细胞色素P3A4、细胞色素P3A5和细胞色素P3A7的mRNA表达水平从2倍增加到3倍。孕烷X受体mRNA在HepG2细胞中表达，而在HFL细胞中不表达。地塞米松诱导的睾酮6β羟基酶活性约为对照的2倍。然而，联合应用RIF并不改变地塞米松诱导的细胞色素P3A基因表达和睾酮6β羟基酶活性。糖皮质激素受体(GR)拮抗剂RU486呈剂量依赖性抑制Dex诱导的细胞色素P3A基因表达。在5μM RU486时，地塞米松诱导的细胞色素P3A4、细胞色素P3A5和细胞色素P3A7mRNA的表达几乎完全被抑制。这些结果表明，在人胎肝细胞中，PXR不参与地塞米松诱导的细胞色素P3A4和细胞色素P3A7的诱导，这种诱导是由GR直接介导的。较少

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