本文選題：膽固醇 + NPC1L1�。� 參考：《新疆醫(yī)科大學》2016年博士論文

【摘要】：目的:(1)識別膽固醇關鍵基因變異與疾病的關聯(lián)研究,為新疆地區(qū)漢族、維族、哈族人群心血管疾病高危人群制定和防止疾病最佳治療方案提供幫助。(2)探討Numb基因多態(tài)性與膽固醇吸收的分子機制研究。(3)探討新疆漢族、維吾爾族人群Numb基因多態(tài)性與冠心病及血脂的相關性研究。方法:(1)收集民族、性別、年齡均匹配的健康患者共349例并根據(jù)低密度脂蛋白-膽固醇水平(LDL-C),分極高、極低組,采用多重酶聯(lián)免疫法PCR的方法,對32條膽固醇相關基因,共635個外顯子片段進行高通量二代測序,分析基因變異位點與血脂的關系。(2)采用Numb基因的所有外顯子和外顯子-內(nèi)含子連接區(qū)域測序的方法,對671例血脂LDL-C極高,極低組患者進行分析,發(fā)現(xiàn)Numb基因新的突變位點,利用細胞實驗對Numb變異位點與膽固醇吸收的分子機制進行研究。(3)采用病例-對照研究,選擇經(jīng)冠狀動脈造影確診的冠心病患者890例和造影陰性的健康人群對照組786例,利用Taqman技術,對Numb基因多態(tài)性進行基因分型以及單體型構(gòu)建,分析Numb基因標簽SNPs和突變位點與冠心病及血脂的關系。結(jié)果:(1)本研究利用人類膽固醇關鍵基因外顯子測序的方法對新疆漢族、維吾爾族、哈薩克族人群進行差異性篩選并篩查獲得膽固醇基因表達差異32條。漢族相較于其他兩個民族獨有的1條差異基因;維吾爾族相較于其他兩個民族獨有的膽固醇相關基因差異有6條;哈薩克族相較于其他兩個民族獨有的1條差異基因,說明民族間基因差異表達,對于防治心血管疾病提供了一定的基礎。(2)對新疆地區(qū)篩選的671例患者中進行Numb基因外顯子測序,發(fā)現(xiàn)基因編碼區(qū)的兩個非同義突變和八個同義突變,兩個非同義突變(A587T和Q533K)均分布在血漿低密度脂蛋白-膽固醇水平極低組。(3)對Numb變異位點進行膽固醇響應、蛋白定位、親和力及穩(wěn)定性等實驗,進一步在細胞水平上驗證兩個突變體在膽固醇循環(huán)中的作用,發(fā)現(xiàn)與野生型相比,Numb A587T和Q533K變異均對膽固醇響應和穩(wěn)定性檢測無明顯差別,而細胞定位較少和相關蛋白親和力的作用減弱。(4)Numb基因標簽SNP的位點(rs2108552)與冠心病的發(fā)病具有相關性。在新疆漢族總體和男性人群中Numb基因rs2108552的顯性模型與冠心病具有關聯(lián)性,在調(diào)整了體質(zhì)指數(shù)(BMI)、甘油三酯(TG)、總膽固醇(TC)、低密度脂蛋白-膽固醇(LDL-C)、高血壓、糖尿病及吸煙等危險因素的影響,rs2108552的顯性模型在總體和男性人群中仍存在差異并有統(tǒng)計學意義(總:OR:1.876,95%CI:1.482~1.979,P=0.004;男:OR:1.498,95%CI:1.305~1.815,P=0.006),而在女性人群中差別無統(tǒng)計學意義。在維吾爾族人群中未發(fā)現(xiàn)此位點與冠心病具有相關性。另外,Numb基因兩個突變位點(A587T和Q533K)在漢族、維吾爾族人群中未發(fā)現(xiàn)與冠心病具有相關性。(5)Numb基因3個標簽SNPs位點在漢族和維吾爾族人群中共建立5個單體型。在漢族總體、男性人群中,G-G-T單體型對照組中的頻率顯著高于CAD組(P=0.007,OR=0.616;P=0.013,OR=0.701),而T-C-T單體型在病例組頻率顯著高于對照組(P=0.002,OR=1.482;P=0.004,OR=1.334),而在女性人群中差別無統(tǒng)計學意義。在維吾爾族人群中,冠心病組和對照組單體型頻率未發(fā)現(xiàn)差別有統(tǒng)計學意義。(6)在調(diào)整性別、年齡、民族、肥胖、吸煙等因素前后rs12435797基因型均與TC水平,rs2108552基因型與TC、LDL-C水平,rs1019075與TC水平具有關聯(lián)性且差別有統(tǒng)計學意義(均P0.05)。另外,Numb基因兩個突變位點(A587T和Q533K)等位基因與TC、LDL-C、HDL-C具有關聯(lián)性且差別有統(tǒng)計學意義(均P=0.000)。結(jié)論:(1)基因外顯子測序結(jié)果表明血脂相關基因在民族間有差異表達變化,特別是維吾爾族和哈薩克族;發(fā)現(xiàn)與膽固醇吸收關鍵基因NPC1L1,Numb,SOAT2及基因突變位點。(2)新發(fā)現(xiàn)的Numb基因的兩個非同義突變A587T和Q533K可能對Numb蛋白膽固醇吸收的功能有影響。(3)Numb基因rs2108552的CC基因型以及三個單核苷酸多態(tài)性的單體型T-C-T可能是漢族男性人群中冠心病發(fā)病的危險因素,G-G-T單體型是冠心病發(fā)病的保護因素。(4)Numb基因3個SNPs基因型和2個突變位點與一個或兩個以上的血脂指標具有關聯(lián)性且突變型的TC和LDL-C水平較低,而HDL-C水平較高。
[Abstract]:Objective: (1) to identify the correlation between the key gene mutation of cholesterol and the disease, and to provide help for the formulation and prevention of the best treatment for the high-risk population of the Han, Uygur and Kazakh people in Xinjiang. (2) to explore the molecular mechanism of Numb gene polymorphism and cholesterol absorption. (3) to explore the Num of the Han and Uygur people in Xinjiang. Study on the correlation between B gene polymorphism and coronary heart disease and blood lipid. Methods: (1) a total of 349 healthy patients with matched national, sex and age were collected and the low density lipoprotein cholesterol level (LDL-C) was very high and very low. The multiple ELISA PCR method was used for 32 cholesterol related genes and a total of 635 exons were high. Two generations of fluxes were sequenced to analyze the relationship between gene mutation sites and blood lipids. (2) the method of sequencing of all exons and exons and introns of the Numb gene was used to analyze 671 patients with extremely high blood lipid LDL-C and extremely low groups, and to find a new mutation site of the Numb gene and use cell experiments to absorb the Numb heterotopic point and cholesterol. Molecular mechanism was studied. (3) a case control study was used to select 890 cases of coronary heart disease diagnosed by coronary angiography and 786 cases of contrast negative healthy population control group. Using Taqman technique, the Numb gene polymorphisms were genotyping and haplotype construction, and Numb gene label SNPs and mutation sites were analyzed with coronary heart disease and blood. Results: (1) in this study, we screened and screened the Han, Uygur and Kazak population of Xinjiang, Uygur and Kazakh people by using the exons of human cholesterol key genes to screen and screen 32 differences in cholesterol gene expression. Compared to the other two ethnic groups, the Han nationality was compared with the other two genes, and the Uygur ethnic group was compared to the other two. There are 6 unique differences in cholesterol related genes in ethnic groups, and Kazak is compared to 1 different genes in other two ethnic groups, indicating the difference in gene expression between ethnic groups, which provides a certain basis for the prevention and control of cardiovascular disease. (2) the Numb gene exon sequencing was carried out in 671 patients screened in Xinjiang, and the gene coding region was found. Two unsynonymous and eight synonymous mutations, two unsynonymous mutations (A587T and Q533K) were distributed in a very low level of plasma low density lipoprotein cholesterol level. (3) the cholesterol response, protein localization, affinity and stability of the Numb heterotopic points were carried out to further verify the two mutant in the cholesterol cycle at the cell level. It was found that the Numb A587T and Q533K variations were not significantly different from those of the wild type, but there was less cell location and associated protein affinity. (4) the locus (rs2108552) of the Numb gene label SNP (rs2108552) was related to the incidence of coronary heart disease. In the population of Xinjiang Han population and the male population, Numb The dominant model of the gene rs2108552 is associated with coronary heart disease, and the dominant model of rs2108552 still exists in the overall and male population and has statistical significance in adjusting the influence of the body mass index (BMI), triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), hypertension, diabetes and smoking. Total: OR:1.876,95%CI:1.482~1.979, P=0.004, men: OR:1.498,95%CI:1.305~1.815, P=0.006), and there is no statistically significant difference in the female population. In Uygur people, this site is not found to be associated with coronary heart disease. In addition, two mutation sites (A587T and Q533K) of the Numb gene are not found in the Uygur population with coronary heart disease. (5) the 3 labelling SNPs loci of the Numb gene set up 5 haplotypes in the Han and Uygur population. In the Han population, the frequency of G-G-T haplotype control group was significantly higher than that of the CAD group (P=0.007, OR=0.616; P=0.013, OR=0.701), and the frequency of T-C-T monotype in the case group was significantly higher than that of the control group (P=0.002, OR=1.482) In the Uygur population, there was no significant difference in haplotype frequency between the CHD group and the control group in the Uygur population. (6) the rs12435797 genotype of the rs12435797 and the TC, the rs2108552 genotypes and TC, the LDL-C level, and the rs101 in the sex, age, nationality, obesity, smoking and other factors were adjusted. 9075 and TC levels were correlated and statistically significant (P0.05). In addition, the two mutation sites (A587T and Q533K) alleles of the Numb gene were associated with TC, LDL-C, and HDL-C (all P=0.000). Conclusion: (1) the results of exons of the gene showed that the blood lipid related genes were differentially expressed among ethnic groups. Especially the Uygur and Kazakh people; found the key genes of cholesterol absorption NPC1L1, Numb, SOAT2 and gene mutation. (2) the two non synonymous mutations of the newly discovered Numb gene, A587T and Q533K, may have an effect on the function of the Numb protein cholesterol absorption. (3) the CC genotypes of the Numb gene rs2108552 and the three single nucleotide polymorphisms Haplotype T-C-T may be a risk factor for coronary heart disease in the Han male population. G-G-T haplotype is a protective factor for coronary heart disease. (4) the 3 SNPs genotypes and 2 mutation sites of the Numb gene are associated with one or more than two blood lipids, and the level of TC and LDL-C is lower and the level of HDL-C is higher.

【學位授予單位】：新疆醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R541.4
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本文編號：1830824