本文選題：FABP1 + 基因多態(tài)性��； 參考：《福建醫(yī)科大學(xué)》2016年碩士論文

【摘要】：近20年以來,隨著社會的進(jìn)步及經(jīng)濟(jì)的發(fā)展,人民生活水平以及生活方式發(fā)生了巨大變化。研究表明,中國人群血脂異常的患病率呈上升趨勢,并趨向年輕化,已成為影響人群健康的重大公共衛(wèi)生問題。血脂異常則是肥胖、心血管疾病以及胰島素抵抗等疾病發(fā)病的主要危險(xiǎn)因素。因此,對影響血脂代謝的因素的研究,探索其可能的發(fā)病機(jī)制,對預(yù)防和控制血脂異常及與之相關(guān)疾病的發(fā)生,提高人群的健康狀態(tài)具有重要的科學(xué)意義。目的:(1)探討福州市漢族健康人群FABP1基因啟動子區(qū)域內(nèi)的單核苷酸多態(tài)性位點(diǎn)rs2919872GA與rs2970903AG的基因型及等位基因頻率分布特征;(2)分析FABP1基因啟動子區(qū)域內(nèi)的rs2919872GA與rs2970903AG對血脂水平的影響;(3)初步探討FABP1基因啟動子區(qū)域rs2919872AG與rs2970903AG的功能。方法:(1)采用橫斷面流行病學(xué)調(diào)查研究方法,以2012年8月至2013年1月在福建醫(yī)科大學(xué)附屬協(xié)和醫(yī)院體檢中心的1182名健康體檢人員為調(diào)查對象,分析FABP1基因啟動子SNPs rs2919872GA、rs2970903AG對總膽固醇(total cholesterol,TC)、甘油三酯(Triglyceride,TG)、低密度脂蛋白膽固醇(Low Density Lipoprotein-Cholesterol,LDL-C)、高密度脂蛋白膽固醇(High Density Lipoprotein-Cholesterol,HDL-C)等指標(biāo)的影響,了解FABP1基因啟動子多態(tài)性與人群血脂水平的關(guān)系。(2)利用融合PCR技術(shù),構(gòu)建FABP1基因啟動子不同單體型的熒光素酶報(bào)告基因重組載體pGL3-rs2919872A和pGL3-rs2970903G,pGL3-rs2919872A/rs2970903G,通過雙熒光素酶表達(dá)檢測系統(tǒng),分析FABP1基因啟動子不同單體型對FABP1轉(zhuǎn)錄活性的影響。(3)采用電泳遷移率實(shí)驗(yàn)研究rs2919872GA等位基因特異探針與HepG2核蛋白結(jié)合能力的差異,并通過生物信息學(xué)預(yù)測FABP1基因啟動子rs2919872GA位點(diǎn)所在序列存在的轉(zhuǎn)錄因子結(jié)合位點(diǎn),分析該位點(diǎn)基因突變是否影響轉(zhuǎn)錄因子的結(jié)合能力,以初步鑒定SNP的功能。結(jié)果:(1)在FABP1基因啟動子區(qū)域的位點(diǎn)rs2919872G等位基因攜帶者(GG+GA基因型)的平均血清甘油三酯水平高于A等位基因攜帶者(AA基因型),差異具有統(tǒng)計(jì)學(xué)意義;多元線性回歸分析結(jié)果也同樣顯示FABP1rs2919872GA是影響TG水平的獨(dú)立因素,rs2919872 A等位基因與TG呈負(fù)相關(guān)。本研究未發(fā)現(xiàn)FABP1 rs2970903AG多態(tài)性與血脂水平及其他臨床指標(biāo)之間存在統(tǒng)計(jì)學(xué)關(guān)聯(lián)。(2)成功構(gòu)建FABP1基因啟動子不同單體型的熒光素酶報(bào)告基因重組載體,包括野生基因型pGL3-2125,和突變基因型pGL3-rs2919872A,pGL3-rs2970903G和pGL3-rs2919872A/rs2970903G;雙熒光素酶活性檢測結(jié)果顯示,在HepG2和Huh7細(xì)胞中,轉(zhuǎn)染pGL3-rs2919872A和pGL3-rs2919872A/rs2970903G突變基因型重組載體相比轉(zhuǎn)染野生基因型重組載體pGL3-2125的相對熒光素酶活性值均降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),而轉(zhuǎn)染pGL3-rs2970903G突變基因型重組載體的相對熒光素酶活性值與野生基因型重組載體p GL3-2125組之間差異無統(tǒng)計(jì)學(xué)意義(P0.05),說明rs2919872A等位基因可以降低FABP1啟動子活性。(3)凝膠遷移阻滯實(shí)驗(yàn)(Electrophoretic mobility shift assay,EMSA)實(shí)驗(yàn)初步鑒定rs2919872GA等位變化改變了與轉(zhuǎn)錄因子結(jié)合的能力。通過生物信息學(xué)預(yù)測發(fā)現(xiàn)原始序列rs2919872G存在一個(gè)GR-alpha轉(zhuǎn)錄因子結(jié)合位點(diǎn),當(dāng)該位點(diǎn)突變?yōu)锳后,則不存在該轉(zhuǎn)錄因子的結(jié)合位點(diǎn),但同時(shí)增加了一個(gè)TFIID轉(zhuǎn)錄因子結(jié)合位點(diǎn)。結(jié)論:(1)通過在中國人群中的流行病調(diào)查研究,發(fā)現(xiàn)FABP1基因啟動子rs2919872A等位基因攜帶者平均血清甘油三酯水平低于G等位基因攜帶者,提示rs2919872GA等位基因的突變可以降低血清甘油三酯水平。(2)通過雙熒光素酶報(bào)告系統(tǒng)及EMSA等實(shí)驗(yàn),初步推測rs2919872GA等位基因的突變改變了該序列與轉(zhuǎn)錄因子結(jié)合的能力,導(dǎo)致FABP1的表達(dá)受到影響,最終引起血清TG水平的改變。(3)為了確定由于基因多態(tài)性存在而導(dǎo)致的潛在轉(zhuǎn)錄因子結(jié)合位點(diǎn)種類的變化,我們還需要通過后續(xù)試驗(yàn)對rs2919872GA的功能進(jìn)行更深入的研究和探討。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R589.2

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