Genetic analysis of cytochrome P4502E1 in gastrointestinal and liver diseases.

细胞色素P4502E1 （2E1）是肝脏非酒精脱氢酶代谢途径中主要的乙醇氧化酶。最近，在人类CYP2E1结构基因的5'-franking区域发现了一种称为PstI/PsaI的限制性片段长度多态性（RFLP）。其中一种CYP2E1等位基因变异，即c2，与野生型c1等位基因相比，在转染到Hep G2细胞后表现出增强的转录活性。然而，目前尚不清楚CYP2E1基因的这些等位基因变异是否会影响人类肝脏中2E1蛋白的表达。因此，我们试图描述CYP2E1基因型与人类肝脏中2E1 mRNA和2E1蛋白表达之间的关系。肝活检样本来自无明显肝病的非酒精受试者。CYP2E1基因型包括A型（c1为纯合子）、B型（c1和c2为杂合子）和C型（c2为纯合子），通过PCR扩增CYP2E1 5'-上游区域，然后用PstI和RsaI酶切扩增的DNA片段。首先将肝脏标本中提取的总RNA进行RT-PCR，然后将扩增的2E1 cDNA与地高辛标记的2E1 cDNA探针在slot blots上杂交。采用人2E1抗体，采用Western blots检测组织活检样品制备的微粒体中2E1蛋白的含量；采用对硝基酚作为代谢探针，检测2E1酶活性。CYP2E1 B基因型受试者的肝脏2E1 mRNA和2E1蛋白表达水平均显著升高。我们的研究结果表明，CYP2E1 PstI/RsaI多态性不仅显著影响人类P450基因的转录，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.