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Genetic analysis of cytochrome P4502E1 in gastrointestinal and liver diseases.

细胞色素P4502E1在胃肠道和肝脏疾病中的遗传分析。

基本信息

批准号：
05670503
负责人：
TSUTSUMI Mikihiro
金额：
$ 1.47万
依托单位：
Kanazawa Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670503/
关键词：
Alcoholic liver disease Cytochrome P4502E1 Genetic polymorphisms Genotypes 食道癌胃癌大腸癌 P4502E1

项目摘要

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.

细胞色素P4502 E1（2 E1）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CYP 2 E1结构基因的5 '-franking区域发现了一种称为Pst I/Psa I的限制性片段长度多态性（RFLP）。一个CYP 2 E1等位基因变异体的特点，即c2，表现出增强的转录活性转染到Hep G2细胞相比，野生型c1等位基因。然而，尚不清楚CYP 2 E1基因的这些等位基因变异是否影响2 E1蛋白在人肝脏中的表达。因此，我们试图描述CYP 2 E1基因型和肝脏表达的2 E1 mRNA和2 E1蛋白在man. Liver活检样本，从非酒精性受试者没有明显的肝脏疾病。CYP 2 E1基因型，包括A型（c1纯合子），B型（c1和c2杂合子）和C型通过PCR扩增CYP 2 E1 5 ′-上游区域，然后用Pst I和Rsa I限制性消化扩增的DNA片段来确定2 E1 mRNA（c2纯合）。通过首先将从肝脏样品中提取的总RNA进行RT-PCR，然后将扩增的2 E1 cDNA在狭缝印迹上与地高辛标记的2 E1 cDNA探针杂交。使用人2 E1抗体通过蛋白质印迹法测量从活检样品制备的微粒体中的2 E1蛋白含量，同时使用对硝基苯酚作为代谢探针评估2 E1酶活性。具有CYP 2 E1 B基因型的受试者表达显著更高水平的肝脏2 E1 mRNA和2 E1蛋白。我们的研究结果表明，CYP 2 E1 PstI/RsaI多态性显着影响不仅这个人P450基因的转录，但也相应的酶在肝脏中表达的水平。