FABP1基因启动子多态性与血脂的关联及功能研究

发布时间：2018-06-25 22:40

本文选题：FABP1 + 基因多态性　；参考：《福建医科大学》2016年硕士论文

【摘要】：近20年以来,随着社会的进步及经济的发展,人民生活水平以及生活方式发生了巨大变化。研究表明,中国人群血脂异常的患病率呈上升趋势,并趋向年轻化,已成为影响人群健康的重大公共卫生问题。血脂异常则是肥胖、心血管疾病以及胰岛素抵抗等疾病发病的主要危险因素。因此,对影响血脂代谢的因素的研究,探索其可能的发病机制,对预防和控制血脂异常及与之相关疾病的发生,提高人群的健康状态具有重要的科学意义。目的:(1)探讨福州市汉族健康人群FABP1基因启动子区域内的单核苷酸多态性位点rs2919872GA与rs2970903AG的基因型及等位基因频率分布特征;(2)分析FABP1基因启动子区域内的rs2919872GA与rs2970903AG对血脂水平的影响;(3)初步探讨FABP1基因启动子区域rs2919872AG与rs2970903AG的功能。方法:(1)采用横断面流行病学调查研究方法,以2012年8月至2013年1月在福建医科大学附属协和医院体检中心的1182名健康体检人员为调查对象,分析FABP1基因启动子SNPs rs2919872GA、rs2970903AG对总胆固醇(total cholesterol,TC)、甘油三酯(Triglyceride,TG)、低密度脂蛋白胆固醇(Low Density Lipoprotein-Cholesterol,LDL-C)、高密度脂蛋白胆固醇(High Density Lipoprotein-Cholesterol,HDL-C)等指标的影响,了解FABP1基因启动子多态性与人群血脂水平的关系。(2)利用融合PCR技术,构建FABP1基因启动子不同单体型的荧光素酶报告基因重组载体pGL3-rs2919872A和pGL3-rs2970903G,pGL3-rs2919872A/rs2970903G,通过双荧光素酶表达检测系统,分析FABP1基因启动子不同单体型对FABP1转录活性的影响。(3)采用电泳迁移率实验研究rs2919872GA等位基因特异探针与HepG2核蛋白结合能力的差异,并通过生物信息学预测FABP1基因启动子rs2919872GA位点所在序列存在的转录因子结合位点,分析该位点基因突变是否影响转录因子的结合能力,以初步鉴定SNP的功能。结果:(1)在FABP1基因启动子区域的位点rs2919872G等位基因携带者(GG+GA基因型)的平均血清甘油三酯水平高于A等位基因携带者(AA基因型),差异具有统计学意义;多元线性回归分析结果也同样显示FABP1rs2919872GA是影响TG水平的独立因素,rs2919872 A等位基因与TG呈负相关。本研究未发现FABP1 rs2970903AG多态性与血脂水平及其他临床指标之间存在统计学关联。(2)成功构建FABP1基因启动子不同单体型的荧光素酶报告基因重组载体,包括野生基因型pGL3-2125,和突变基因型pGL3-rs2919872A,pGL3-rs2970903G和pGL3-rs2919872A/rs2970903G;双荧光素酶活性检测结果显示,在HepG2和Huh7细胞中,转染pGL3-rs2919872A和pGL3-rs2919872A/rs2970903G突变基因型重组载体相比转染野生基因型重组载体pGL3-2125的相对荧光素酶活性值均降低,差异具有统计学意义(P0.05),而转染pGL3-rs2970903G突变基因型重组载体的相对荧光素酶活性值与野生基因型重组载体p GL3-2125组之间差异无统计学意义(P0.05),说明rs2919872A等位基因可以降低FABP1启动子活性。(3)凝胶迁移阻滞实验(Electrophoretic mobility shift assay,EMSA)实验初步鉴定rs2919872GA等位变化改变了与转录因子结合的能力。通过生物信息学预测发现原始序列rs2919872G存在一个GR-alpha转录因子结合位点,当该位点突变为A后,则不存在该转录因子的结合位点,但同时增加了一个TFIID转录因子结合位点。结论:(1)通过在中国人群中的流行病调查研究,发现FABP1基因启动子rs2919872A等位基因携带者平均血清甘油三酯水平低于G等位基因携带者,提示rs2919872GA等位基因的突变可以降低血清甘油三酯水平。(2)通过双荧光素酶报告系统及EMSA等实验,初步推测rs2919872GA等位基因的突变改变了该序列与转录因子结合的能力,导致FABP1的表达受到影响,最终引起血清TG水平的改变。(3)为了确定由于基因多态性存在而导致的潜在转录因子结合位点种类的变化,我们还需要通过后续试验对rs2919872GA的功能进行更深入的研究和探讨。
[Abstract]:In the last 20 years, with the progress of society and the development of economy, the living standard and lifestyle of the people have changed greatly. The study shows that the prevalence rate of blood lipid abnormality in the Chinese population is on the rise, and tends to be young, and has become a major public health problem affecting the health of the population. The main risk factors of the disease such as insulin resistance, therefore, to study the factors affecting the metabolism of blood lipids, explore the possible pathogenesis, to prevent and control the abnormal blood lipid and related diseases, and to improve the health of the population. Objective: (1) to explore the FABP1 base of the healthy population of the Han nationality in Fuzhou The genotype and allele frequency distribution of single nucleotide polymorphic loci rs2919872GA and rs2970903AG in the promoter region; (2) the analysis of the effect of rs2919872GA and rs2970903AG on the level of blood lipid in the FABP1 promoter region; (3) the function of rs2919872AG and rs2970903AG in the FABP1 gene promoter region. Method: (1) A cross-sectional epidemiological survey was conducted to analyze the FABP1 gene promoter SNPs rs2919872GA, rs2970903AG to total cholesterol (total cholesterol, TC), triglyceride (Triglyceride, TG), and low density, from August 2012 to January 2013 at the medical check-up center of the Affiliated Union Hospital of Fujian Medical University. The influence of Low Density Lipoprotein-Cholesterol (LDL-C), high density lipoprotein cholesterol (High Density Lipoprotein-Cholesterol, HDL-C) and so on, to understand the relationship between the polymorphism of the FABP1 gene promoter and the blood lipid level of the population. (2) using the fusion PCR technology to construct the different haplotype fluorescein of the FABP1 gene promoter. Enzyme reporter gene recombinant vector pGL3-rs2919872A and pGL3-rs2970903G, pGL3-rs2919872A/rs2970903G, through the dual luciferase expression detection system, the effect of different haplotypes of FABP1 promoter on FABP1 transcriptional activity was analyzed. (3) the binding ability of rs2919872GA allele specific probes and HepG2 nucleoprotein was studied by electrophoretic mobility test. The difference, and the prediction of the binding site of the transcriptional factor in the sequence of the rs2919872GA loci of the FABP1 gene by bioinformatics, and the analysis of whether the mutation of the site affects the binding capacity of the transcription factor to identify the function of the SNP. Results: (1) the rs2919872G allele carrier of the locus of the FABP1 gene promoter region (G) The average serum triglyceride level of the G+GA genotype was higher than that of the A allele carrier (AA genotype), and the difference was statistically significant. The results of multiple linear regression analysis also showed that FABP1rs2919872GA was an independent factor affecting the TG level, and the rs2919872 A allele was negatively correlated with TG. The polymorphism of FABP1 rs2970903AG was not found in this study. There was a statistical correlation between the blood lipid level and other clinical indicators. (2) the recombinant vector of the luciferase reporter gene of the FABP1 gene promoter was successfully constructed, including the wild genotypic pGL3-2125, and the mutant genotype pGL3-rs2919872A, pGL3-rs2970903G and pGL3-rs2919872A/rs2970903G; the results of the double luciferase activity detection showed a significant result. In HepG2 and Huh7 cells, the relative luciferase activity values of the transfected pGL3-rs2919872A and pGL3-rs2919872A/rs2970903G recombinant vectors were lower than those of the transfected wild genotypic recombinant vector pGL3-2125, and the difference was statistically significant (P0.05), while the relative fluorescein transfected with the recombinant vector of the pGL3-rs2970903G mutant genotype was relative to fluorescein. There was no significant difference between the enzyme activity value and the P GL3-2125 group (P0.05), indicating that the rs2919872A allele could reduce the activity of FABP1 promoter. (3) the gel migration block experiment (Electrophoretic mobility shift assay, EMSA) preliminarily identified that the rs2919872GA allele changes were associated with the transcription factors. Ability. Through bioinformatics prediction, it was found that there was a GR-alpha transcription factor binding site in the original sequence rs2919872G. When the site mutation was A, there was no binding site of the transcription factor, but a TFIID transcription factor binding site was added. Conclusion: (1) through the epidemiological investigation in the Chinese population, the discovery of FABP The average serum triglyceride level of the 1 gene promoter of the 1 gene promoter was lower than that of the G allele carriers, suggesting that the mutation of the rs2919872GA allele could reduce the level of serum triglyceride. (2) the mutation of the rs2919872GA allele changed the sequence by the double Luciferase Report System and the EMSA experiment. The ability of transcription factor binding leads to the influence of FABP1 expression and ultimately changes the level of serum TG. (3) in order to determine the variety of potential transcription factor binding sites caused by genetic polymorphism, we also need to further study and explore the function of rs2919872GA through follow-up tests.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R589.2

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