Stable expression of human cytochrome P450 2D6*10 in HepG2 cells

作     者：JianZhuge Ying-NianYu Xiao-DanWu 

作者机构：DepartmentofPathologyandPathophysiologySchoolofMedicineZhejiangUniversityHangzhou310031ZhejiangProvinceChina CenterofAnalysisZhejiangUniversityHangzhou310031ZhejiangProvinceChina 

出 版 物：《World Journal of Gastroenterology》 (世界胃肠病学杂志（英文版）)

年 卷 期：2004年第10卷第2期

页      面：234-237页

核心收录：

学科分类：0831[工学-生物医学工程（可授工学、理学、医学学位）] 08[工学] 

基　　金：Supported by National Natural Science Foundation of China No.39770868 and Natural Science Foundation of Zhejiang Province No.397490 

主　　题：P-450细胞色素 HepG2细胞 逆转录聚合酶链反应 高效液相色谱法 肝脏同功酶 DNA序列 

摘      要：AIM:Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver *** most important enzymes are CYPIA2, 3A4,2C9/19,2D6 and *** CYP2D6 accounts for2% of the total CYP liver enzyme content,it mediates metabolism in almost 25% of *** order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was ***:Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from human liver tissue and cloned into pGEM-T vector,cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9.A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 *** of mRNA was validated by *** activity of catalyzing dextromethorphan Odemethylation in postmitochondrial supernant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC).RESULTS:The cloned cDNA had 4 base differences, e.g.100 C→T, 336 T→C,408 C→G and 1 457 G→C,which resulted in P34S,and S486T amino acid substitutions, and two samesense mutations were 112F and 136V compared with that reported by Kimura et al (GenBank accession number: M33388).P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 *** relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31±0.19nmol·min^-1·mg^-1 S9 protein (n=3),but was undetectable in parental HepG2 ***:cDNA of human CYP2D6*IOcan be successfully doned.A cell line,HepG2-CYP2D6*10,expressing CYP2D6*10 mRNA and having metabolic activity,has been established.