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

再生医学基础研究：人胎儿肝细胞的分化及其在药物药代动力学实验中的应用

• 批准号: 16590109
• 负责人: MATSUNAGA Tamihide
• 金额: $ 2.37万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590109/
• 关键词: human fetal liver cell; cytochrome P450; dexamethasone; glucocorticoid receptor; CYP3A4; CYP3A7; rifampicin; RU486; 正常ヒト胎児肝細胞; オンコスタチンM; グルココルチコイド受容; メチル化; サイトケラチン7; 移植; 分化; RU-486

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

人胎肝（HFL）细胞培养物从六个正常人肝组织的库开始。用3-[4，5-二甲基噻唑-2-基]-2，5-二苯基四氮唑溴化物测定HFL细胞的增殖和活力，通过培养15天，细胞增加超过100倍。随着培养时间的延长，白蛋白（ALB）、肝细胞生长因子4 α（HGF）、肝细胞生长因子（HGF）、CYP 3A 4、CYP 3A 5和CYP 3A 7的mRNA表达水平逐渐升高，甲胎蛋白（AFP）mRNA表达水平逐渐降低。而在HepG 2细胞中，ALB和AFP mRNA的表达水平没有变化，而CYP 3A 4、CYP 3A 5和CYP 3A 7 mRNA的表达水平逐渐下降。包括CYP 3A在内的主要CYP亚型的mRNA表达，在HepG 2细胞中可检测到CYP 1A 2、CYP 2A 6、CYP 2B 6、CYP 2C（2C 9和2C 19）、CYP 2D 6和CYP 2 E1。除CYP 1A 2外，HepG 2细胞中表达的所有CYP 1A 2 mRNA在HFL细胞中均被检测到。在HFL细胞中，CYP 3A ...更多信息 地塞米松（DEX）能显著上调CYP 4 mRNA和CYP 3A 7 mRNA的表达，而利福平（RIF）对CYP 4 mRNA和CYP 3A 7 mRNA的表达无明显影响。DEX、RIF或苯巴比妥（PB）均未显著增加CYP 3A 5 mRNA水平。在另一方面，CYP 3A 4，CYP 3A 5，和CYP 3A 7 mRNA表达水平在HepG 2细胞增加2- 3倍，用DEX，PB，和RIF处理。孕烷X受体mRNA在HepG 2细胞中有表达，而在HFL细胞中无表达。DEX诱导的TEX 6β -羟化酶活性约为对照的2倍。然而，与RIF联合治疗未改变DEX介导的CYP 3A mRNA表达和睾酮6β-羟化酶活性诱导。糖皮质激素受体（GR）拮抗剂RU 486以剂量依赖性方式抑制DEX介导的CYP 3A mRNA诱导。在5 μM RU 486时，DEX介导的CYP 3A 4、CYP 3A 5和CYP 3A 7 mRNA表达诱导几乎完全受到抑制。这些结果表明，在人胎肝细胞中，PXR不参与DEX介导的CYP 3A 4和CYP 3A 7的诱导，并且诱导直接由GR介导。

项目成果

Expressions of cytochrome P450 in differentiating mouse embryonic stem cells.

细胞色素P450在分化小鼠胚胎干细胞中的表达。

• 发表时间: 2005
• 期刊: Drug Metab Rev 37
• 作者: Matsunaga T;Kose E;Yasuda S;Ise H;Ikeda U;Ohmori S;松永民秀;Matsunaga T et al.;Maruyama M et al.;Ohmori S et al.;Matsunaga T et al.
• 通讯作者: Matsunaga T et al.

Effective NADH-dependent oxidation of 7β-hydroxy-Δ8-tetrahydrocannabinol to the corresponding ketone by Japanese monkey hepatic microsomes

• DOI: 10.1248/bpb.28.646
• 发表时间: 2005-04-01
• 期刊: BIOLOGICAL & PHARMACEUTICAL BULLETIN
• 影响因子: 2
• 作者: Matsunaga, T;Higuchi, S;Yamamoto, I
• 通讯作者: Yamamoto, I

Simple and highly sensitive method for determination of P-glycoprotein ATPase activity using Luciferase.

使用荧光素酶测定 P-糖蛋白 ATP 酶活性的简单且高度灵敏的方法。

• 发表时间: 2005
• 期刊: Drug Metab Rev 37
• 作者: Matsunaga T;Kose E;Yasuda S;Ise H;Ikeda U;Ohmori S;松永民秀;Matsunaga T et al.;Maruyama M et al.;Ohmori S et al.
• 通讯作者: Ohmori S et al.

Expression and induction of CYP3As in human fetal hepatocytes

• DOI: 10.1016/j.bbrc.2004.04.041
• 发表时间: 2004-05-28
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Matsunaga, T;Maruyama, M;Ohmori, S
• 通讯作者: Ohmori, S

Induction mechanisms of CYP3As in human fetal hepatocytes by glucocorticoids.

糖皮质激素在人胎儿肝细胞中诱导 CYP3As 的机制。

• 发表时间: 2005
• 期刊: Drug Metab Rev 37
• 作者: Matsunaga T;Kose E;Yasuda S;Ise H;Ikeda U;Ohmori S;松永民秀;Matsunaga T et al.;Maruyama M et al.
• 通讯作者: Maruyama M et al.