Zhuge Jian, Yu Ying-Nian, Wu Xiao-Dan
Department of Pathology and Pathophysiology, School of Medicine, Zhejiang University, Hangzhou 310031, Zhejiang Province, China.
World J Gastroenterol. 2004 Jan 15;10(2):234-7. doi: 10.3748/wjg.v10.i2.234.
Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established.
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 cells. Expression of mRNA was validated by RT-PCR. Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernatant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC).
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 112 F and 136 V compared with that reported by Kimura et al (GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D610 allele. The relative activity of S9 fraction of HepG2-CYP2D610 metabolized detromethorphan O-demethylation was found to be 2.31 +/- 0.19 nmol/min(-1)/mg(-1) S9 protein (n=3), but was undetectable in parental HepG2 cells.
cDNA of human CYP2D610 can be successfully cloned. A cell line, HepG2-CYP2D610, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.
超过90%的药物由肝脏同工酶细胞色素P - 450（CYP）家族代谢。最重要的酶是CYP1A2、3A4、2C9/19、2D6和2E1。尽管CYP2D6占肝脏CYP总酶含量的不到2%，但它介导了近25%的药物代谢。为了研究其药物代谢的酶活性，克隆了其cDNA并建立了稳定表达CYP2D6的HepG2细胞系。
采用逆转录-聚合酶链反应（RT-PCR）从人肝组织提取的总RNA中扩增人CYP2D6 cDNA，并克隆到pGEM-T载体中。通过DNA测序鉴定cDNA片段，并亚克隆到哺乳动物表达载体pREP9中。将pREP9-CYP2D6重组质粒转染至肝癌HepG2细胞中建立细胞系。通过RT-PCR验证mRNA的表达。采用高效液相色谱（HPLC）测定细胞线粒体后上清液（S9）组分中催化右美沙芬O-去甲基化的酶活性。
克隆的cDNA有4个碱基差异，即100 C-T、336 T-C、408 C-G和1457 G-C，导致P34S和S486T氨基酸替换，与Kimura等人报道的（GenBank登录号：M33388）相比，有两个同义突变112 F和136 V。P34S和S486T氨基酸替换是CYP2D610等位基因的特征。发现HepG2-CYP2D610的S9组分代谢右美沙芬O-去甲基化的相对活性为2.31±0.19 nmol/min（-1）/mg（-1）S9蛋白（n = 3），但在亲本HepG2细胞中未检测到。
人CYP2D610的cDNA可成功克隆。已建立表达CYP2D610 mRNA并具有代谢活性的细胞系HepG2-CYP2D6*10。
