Genetic analysis of cytochrome P4502E1 in gastrointestinal and liver diseases.

细胞色素P4502E1(2E1)是肝脏非乙醇脱氢酶代谢途径中的主要乙醇氧化酶。最近，在人类细胞色素P450 2 E1结构基因的5‘-印章区域发现了一种称为PstI/PsaI的限制性片段长度多态性(RFLP)。与野生型的c1等位基因相比，其中一种具有特征的等位基因c2在导入Hep G2细胞后表现出更强的转录活性。然而，目前尚不清楚这些等位基因变异是否会影响2E1蛋白在人肝脏中的表达。因此，我们试图描述人类肝脏2E1mRNA和2E1蛋白表达与细胞色素P4502E1型之间的关系。肝活检样本取自无明显肝病的非酒精性受试者。用聚合酶链式反应(PCR)扩增CYP2E15‘-上游区，然后用PstI和RsaI限制性内切酶对扩增出的DNA片段进行限制性内切酶消化，确定C型(c1和c2纯合子)、B型(c1和c2杂合子)和C型(c2纯合子)。2E1mRNA的定量方法是先将从肝脏提取的总RNA进行RT-PCR，然后将扩增出的2E1cDNAs与地高辛标记的2E1cDNA探针杂交。2以人2E1抗体为底物，采用免疫印迹法检测活检标本微粒体中的2E1蛋白含量；以对硝基苯酚为代谢探针，测定2E1酶活性。携带CYP2E1B基因的受试者肝脏2E1mRNA和2E1蛋白的表达水平均显著高于对照组。我们的结果表明，CYP2E1PstI/RsaI多态不仅显著影响这个人类P450基因的转录，而且还影响相应酶在肝脏中的表达水平。

Cytochrome P4502E1 (2E1) is a major ethanol-oxidizing enzyme of the non-alcohol dehydrogenase metabolic pathway in liver. Recently, a restriction fragment length polymorphism (RFLP) termed PstI/PsaI was found within the 5'-franking region of the human CYP2E1 structural gene. One of the CYP2E1 allelic variants characterized, namely c2, exhibited enhanced transcriptional activity upon transfection into Hep G2 cells when compared to the wild-type c1 allele. However, it is not known whether these allelic variations of the CYP2E1 gene influence the expression of 2E1 protein in human liver. Thus, we sought to describe the relationship between CYP2E1 genotype and hepatic expression of both 2E1 mRNA and 2E1 protein in man. Liver biopsy samples were obtained from non-alcoholic subjects without overt liver disease. CYP2E1 genotypes, including type A (homozygous for c1) , type B (heterozygous for c1 and c2) , and type C (homozygous for c2) , were determined by PCR amplification of the CYP2E1 5'-upstream region, followed by restriction digestion of the amplified DNA fragment with PstI and RsaI.2E1 mRNA was quantified by first subjecting total RNA extracted from the liver specimens to RT-PCR,and then hybridizing the amplified 2E1 cDNAs on slot blots with a digoxigenin-labeled 2E1 cDNA probe. 2E1 protein content in microsomes prepared from the biopsy samples was measured by Western blots using human 2E1 antibody while 2E1 enzyme activity was assesed using p-nitrophenol as a metabolic probe. Subjects with the CYP2E1 B genotype express significantly higher levels of both hepatic 2E1 mRNA and 2E1 protein. Our results indicate that the CYP2E1 PstI/RsaI polymorphism markedly influences not only the transcription of this human P450 gene but also the levels at which the corresponding enzyme is expressed in liver.