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Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (5): 530-537    DOI: 10.3785/j.issn.1008-9292.2013.05.009
    
Characteristics of microsomal phase Ⅱ metabolic enzymes in mouse embryonic stem cell-derived liver tissue
LI Tong1, GUO Mei-Yuan1, MA Kui-Fen1,2, DU Yue1,  He-Liang-Yan1, ZHU Dan-Yan1, LOU Yi-Jia1
1.Institute of Pharmacology,Toxicology and Biochemical Pharmaceutics,College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058,China;2.The First Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310003,China;
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Abstract  

Objective: To investigate the characteristics of phaseⅡ metabolic enzymes in mouse embryonic stem (ES) cell-derived liver tissue.
Methods: Mature hepatocytes were differentiated from embryonic stem cells in cultured mouse embryoid bodies (EB) at d18.Western blot was used to detect the expression of uridine 5′-diphosphate glucronosyl transferase (UGT1a1,UGT1a6) and microsomal glutathione S-transferases 1(mGST1) during the differentiation course.The derived liver tissue was incubated with UDPGA and 7-HFC,the formation of 7-HFC glucuronide was detected by HPLC to examine the total activities of UGT1a1and UGT1a6.Furthermore,the microsomes were incubated with CDNB and GSH,and the mGST1 activity was measured by spectrometry.
Results: An increase tendency of UGT1a1 expression was noticed during the differentiation course.UGT1a6 and mGST1 were not detected in the earlier stage until d18 of differentiation.The metabolic activity of mGST1 in the derived hepatocytes was 7.65 nmol/min/mg on d18.
Conclusion: The ES cell-derived liver tissue possesses partial metabolic function of phase Ⅱenzymes on d18 of differentiation,which might be used as a model for in vitro research on hepatic pathophysiology and phase Ⅱ drug metabolism.



Key wordsEmbryonic stem cells      Liver/anatomy &      histology      Glutathione transferase      Microsomes, Liver      Glucuronosyltransferase     
Received: 21 November 2012      Published: 25 September 2013
CLC:  R 742.1  
Cite this article:

LI Tong, GUO Mei-Yuan, MA Kui-Fen, DU Yue, He-Liang-Yan, ZHU Dan-Yan, LOU Yi-Jia. Characteristics of microsomal phase Ⅱ metabolic enzymes in mouse embryonic stem cell-derived liver tissue. Journal of ZheJiang University(Medical Science), 2013, 42(5): 530-537.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2013.05.009     OR     http://www.zjujournals.com/med/Y2013/V42/I5/530


小鼠胚胎干细胞衍生的肝组织微粒体Ⅱ相代谢酶表征

目的:观察小鼠胚胎干细胞定向分化肝组织过程中,肝组织微粒体Ⅱ相代谢酶尿苷-5′-二磷酸葡醛酰转移酶(UGT)1a1、1a6和微粒体谷胱甘肽S-转移酶1(mGST1)的表达及其催化活性。
方法:利用拟胚体固有的三胚层结构,直接由中胚层心肌细胞产生成纤维细胞生长因子,自发诱导内胚层细胞发育,并由同步分化的内皮细胞支持肝细胞发育形成肝样组织。在不同分化阶段,基于mRNA表达情况,以Western blot法检测UGT1a1、UGT1a6和mGST1表达特征。至分化终点(第18天)以心肌细胞搏动区域附近表达肝特异性标记物白蛋白确定为肝细胞。超声法碎裂细胞,超速离心制备肝细胞微粒体,以HPLC检测UGTs代谢活性,以色谱法测定并计算mGST1酶催化活性。
结果:至分化终点,小鼠胚胎干细胞衍生的肝组织表达UGT1a1、UGT1a6和mGST1,并具有UGT1a1、UGT1a6和mGST1的催化功能。其中分化第18天肝组织GST1催化活性为7.65 nmol·min-1·mg-1
结论:小鼠胚胎干细胞分化的肝组织具UGT1a1、UGT1a6和mGST1代谢功能,可望成为肝脏病理生理和药物Ⅱ相代谢研究的体外模型。


关键词: 胚胎干细胞,  肝/解剖学和组织学,  谷胱甘肽转移酶,  微粒体,肝,  葡糖醛酸基转移酶 
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