Catalase基因多态的研究与功能初探

【作者】 李艳平；

【导师】 金力；

【作者基本信息】 复旦大学 ， 遗传学， 2005， 博士

【摘要】 Catalase蛋白是人体内非常重要的过氧化氢酶,由于其抗氧化的生理功能成为心血管疾病研究的候选基因。我们在大别山隔离群体（324个体）第一次发现catalase启动子区域SNP-844C/T多态与原发性高血压高度相关。考虑到隔离群体遗传背景的相对单纯性,我们继续在上海一般群体的926例个体（病人448例,正常人有478例）进行病例一对照研究,发现SNP-844C/T与原发性高血压表型仍然存在显著相关,这种相关性和年龄可能有一定的关联。进一步证实在隔离人群中分析高血压相关基因是有效的。 为了探索catalase启动子区域多态与catalase表达和血压的关系,我们进行了SNP-844C/T多态的功能研究。构建了包含T和C多态的启动子区域1.14Kb序列的载体,经细胞株转染后进行报告基因荧光素酶检测。HepG2细胞株转染后荧光素酶检测,含C变异比T变异使表达增强22%;ECV-304,293和HeLa细胞株转染后,T比C使荧光素酶表达分别增加47.5%,132%和56.58%。HepG2细胞株没有AP-2蛋白的表达,上述另外三个细胞株有AP-2蛋白表达。Gel shift和supershift实验结果说明C变异和AP-2蛋白结合较强而T变异很弱或者无。Western Blot结果表明血液中catalase蛋白量随基因型由C/C向T/C和T/T变化而呈现降低的趋势。 由以上的结果我们推测AP-2可能是抑制蛋白,在血液可能存在Cat基因的正调控因子。血液对过氧化物的处理能力与血管内皮细胞正常功能有关,后者对血压有贡献。血管内皮细胞产生的H₂O₂能够被血液中catalase所调控,catalase蛋白对于血管内皮细胞氧化状态平衡的维持有着非常重要的作用。我们推测当为C/C基因型时,血液中catalase使内皮细胞氧化还原状态维持平衡状态;当为T/T基因型时血液catalase表达较低,造成血管壁血管内皮细胞氧化还原状态的失衡而最终导致血管内皮细胞受到损伤,最终造成慢性炎症导致血管疾病。这些推测有待于分子生物学实验的进一步验证。对catalase基因深入研究有助于我们对心血管病机制的深入理解。 对中国各地区各民族系统人群的普查发现catalase SNP-844T频率在从广西进入中国的现代人系统（侗傣、阿尔泰等）中偏高,在从云南进入中国的人群（汉藏、苗瑶、南亚）中偏低,存在明显的民族系统性规律,而不存在地理分布规律。更多还原

【Abstract】 Catalase is an important antioxidant enzyme and is chosen as a candidate gene for essential hypertension and other cardiovascular diseases by its physiological role of antioxidation. We have identified a SNP-the catalase gene SNP-844C/T, which is strongly associated with essential hypertension after the study of isolated population of Dabie Mountain in Anhui province. Considering the genetic homogeneity of isolated population, we further carried on the case-control studies among the outbred population composed of 926 individuals from shanghai area （affected,448 subjects; control,478 subjects） .The results confirmed the association between genotype, allele frequencies and phenotype. The association is more evident after age stratification. The above data proved that it is effective to analyze hypertension associated genes in isolated population.We further studied the influence of variation of SNP-844 C/T on catalase expression and blood. The expression of reporter gene luciferase is analyzed in several cell lines transfected with construct vectors containing 1.14Kb catalase upstream sequence with either C or T variant .It is observed that the expression of luciferase increase by 22% with C variant than T variant in HepG2, while the expressions of luciferase increased by 47.5%, 132% and 56.58% separately transfected with T variant compared with C variant in ECV-304, 293 ,and HeLa cell lines. HepG2 is AP-2 deficient and AP-2 protein is expressed in other three cell lines. The results of gel shift and supershift assays showed that C variant could bind AP-2 protein strongly while the T variant demonstrated poorly as the binding activity is concerned. The results of western blot demonstrated an increase trend of catalase protein from T/T, T/C to C/C genotypes in blood .From above data, AP-2 is postulated to be a repressor of catalase. And other transcription factors as potential enhancer is predicted to exist in blood while not in endothelial. The normal function of endothelial is related to the ability of disposal of oxidants by blood microenviroment. And catalase can dispose H₂O₂ released from vascular endothelial which is important in the balance of redox metabolism in endothelial cell. We propose that the redox metabolism of endothelial is balanced under C/C genotype. However the更多还原