脂联素及其受体Poly-miRTS与冠心病相关性研究

发布时间：2018-03-24 21:45

本文选题：单核苷酸多态性　切入点：冠心病　出处：《延边大学》2015年硕士论文

【摘要】：目的：在中国延边地区人群中寻找一个冠心病(CAD)易感性标记物。方法：本研究应用计算机生物信息学预测软件和NCBI-SNP数据库,从脂联素及其受体基因中筛选出和miRNA结合能差异(|△△G|)大于5.27 kcal/mol的6个野生型和变异型等位基因。采用聚合酶链反应-限制性片段长度多态法(PCR-RFLP)方法对6个野生型和变异型等位基因进行基因分型,比较基因型的分布频率,观察与冠心病易感性的相关性。应用PCR扩增3UTR基因组DNA,利用限制性内切酶切断PCR产物及pGL3系列的质粒,利用T4DNA连接酶连接两种DNA,制备pGL3-3'UTR系列的质粒,在E.coli中大量培养后与miRNA表达质粒一起转染给人平滑肌细胞株培养,进行荧光素酶检测,用含海肾萤光素酶(Renilla luciferase)的pRL-SV40质粒作为内部对照物,利用双荧光素酶基因检测系统测定启动子活性.,利用Trizol法提取不同基因型RNA后反转录为cDNA,利用qRT-PCR方法比较不同基因型的mRNA表达量。采用Western-blot法检测结果显示不同基因型的ADIPOR1蛋白表达量。结果：1.ADIPORl-rs7539542C等位基因冠心病发病率显著高于其他等位基因。2.通过用荧光素酶检测测定启动子活性,结果显示只有ADIPORl-rs7539542C有显著性统计学意义,p为0.03。3.qRT-PCR方法显示,rs7539542G时mRNA表达量显著低于rs7539542C,具有统计学意义,p为0.02。4. Western-blot法检测结果显示,rs7539542G时mRNA表达量显著低于rs7539542C,具有统计学意义,p为0.01。结论：本研究结果显示延边地区人群冠心病易感基因,ADIPORl-rs7539542C可作为延边地区预测冠心病易感性标志物。
[Abstract]:Objective: to search for a susceptible marker of coronary artery disease (CAD) in the population of Yanbian area, China. Methods: the computer bioinformatics prediction software and NCBI-SNP database were used in this study. Six wild and variant alleles with miRNA binding energy (G) greater than 5.27 kcal/mol were screened from adiponectin and its receptor genes. Genetic and variant alleles were genotyped. To compare the frequency of genotype distribution and to observe the correlation between genotype distribution and susceptibility to coronary heart disease (CHD), PCR was used to amplify 3UTR genomic DNA, restriction endonuclease was used to cut off PCR products and plasmids of pGL3 series, T4DNA ligase was used to connect the two kinds of plasmids, and plasmids of pGL3-3'UTR series were prepared. Human smooth muscle cell lines were transfected with miRNA expression plasmid in large quantities in E.coli. Luciferase was detected by luciferase assay. The pRL-SV40 plasmid containing luciferase was used as internal control. The promoter activity was determined by double luciferase gene detection system, the RNA of different genotypes was extracted by Trizol method, and the mRNA expression of different genotypes was compared by qRT-PCR method. The results of Western-blot detection showed different bases. Results: 1. The incidence of coronary heart disease of ADIPORl-rs7539542C allele was significantly higher than that of other alleles .2.The promoter activity was determined by luciferase assay. The results showed that the expression of mRNA was significantly lower than that of rs7539542G by 0.03.3.qRT-PCR method, and was significantly lower than that of rs7539542G by 0.03.3.qRT-PCR method. The results of Western-blot assay showed that the expression of mRNA was significantly lower than that of rs7539542G, and the results of Western-blot showed that the expression of mRNA was significantly lower than that of rs7539542G. Conclusion: the results showed that ADIPORl-rs7539542C could be used as a marker to predict the susceptibility of coronary heart disease in Yanbian area.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.4

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