炎症因子基因DNA甲基化与原发性高血压的关联性研究
发布时间：2018-09-12 07:01
【摘要】：目的:1、探讨原发性高血压患者与正常对照间炎症因子基因TLR2、IL6和IFN-γ启动子DNA甲基化水平的差异;2、研究性别、吸烟和饮酒等环境因素对炎症因子基因DNA甲基化差异的影响;3、探讨炎症因子基因启动子Cp G位点的DNA甲基化差异对原发性高血压的临床诊断价值。方法:1、运用多阶段抽样的方法纳入研究对象,进行人口学特征、环境因素的问卷调查、体格检查及实验室检查,最终确定按性别、年龄1:1匹配的96个新发病例和96个对照,并获得192个研究对象的基线资料、血液样本及血生化指标;2、将采集的血样进行DNA的提取、亚硫酸盐修饰以及运用焦磷酸测序法检测血液样本中炎症因子基因TLR2、IL6和IFN-γ启动子DNA甲基化水平;3、通过分别比较检测的炎症因子基因在两组间的甲基化程度,分析甲基化程度的差异,了解环境因素对甲基化及原发性高血压发病的影响,并用Logistic回归校正混杂因素。结果:1、各基因甲基化水平比较:(1)TLR2基因:新发病例组与正常对照组相比,新发病例组TLR2启动子Cp G6位点甲基化水平降低(对照组VS新发病例组(%):Cp G6:8.30±4.13 VS 3.58±3.64,OR(95%CI)=0.91(0.861~0.979),adjusted P=0.009);(2)IL6基因:新发病例组与正常对照组相比,新发病例组IL6启动子Cp G3位点甲基化水平降低(对照组VS新发病例组(%):Cp G3:57.45±8.29VS 51.52±6.18,OR(95%CI)=0.90(0.838~0.965),adjusted P=0.004);(3)IFN-γ基因:新发病例组与正常对照组相比,新发病例组IFN-γ启动子Cp G2位点甲基化水平降低(对照组VS新发病例组(%):Cp G2:10.50±0.92VS 9.23±0.83,OR(95%CI)=0.64(0.480~0.856),adjusted P=0.003)。2、对照组中性别间比较:(1)TLR2基因:女性TLR2启动子Cp G6位点甲基化水平偏高(男性VS女性(%):Cp G6:7.31±1.49 VS 8.94±5.09,adjusted P=0.013);(2)IL6基因:女性IL6启动子Cp G2位点甲基化水平偏低(男性VS女性(%):Cp G2:65.53±7.31 VS 60.26±10.46,adjusted P=0.046);(3)IFN-γ基因:符号秩和检验分析发现,女性IFN-γ启动子Cp G1位点甲基化水平偏低(男性VS女性(%):Cp G1:37.58±1.31 VS 36.52±1.64,adjusted P=0.020)。3、男性中,吸烟、饮酒与炎症因子基因DNA甲基化关联分析:(1)IL6基因:男性中,吸烟者的IL6启动子Cp G2-3甲基化水平降低(不吸烟者VS吸烟者(%):Cp G2:64.28±6.36 VS 60.41±7.74,adjusted P=0.023;Cp G3:57.78±7.87 VS 53.70±8.62,adjusted P=0.038);饮酒者的IL6启动子Cp G2-3甲基化水平降低(不饮酒者VS饮酒者(%):Cp G2:64.70±7.03 VS 60.89±7.32,adjusted P=0.038;Cp G3:60.48±7.58 VS 53.23±7.99,adjusted P=4.24×10-4);(2)TLR2和IFN-γ基因:未发现差异化位点。4、相关性分析:(1)TLR2基因:TLR2启动子Cp G6位点甲基化水平与血压成负相关(Cp G6 and SBP:r=-0.329,adjusted P=3.95×10-5;Cp G6 and DBP:r=-0.304,adjusted P=1.80×10-4);(2)IL6基因:IL6启动子Cp G2-3位点甲基化水平与血压成负相关(Cp G2and SBP:r=-0.274,adjusted P=0.009;Cp G2 and DBP:r=-0.183,adjusted P=0.011;Cp G3 and SBP:r=-0.321,adjusted P=5.60×10-6;Cp G3 and DBP:r=-0.274,adjusted P=1.17×10-4);(3)IFN-γ基因:IFN-γ启动子Cp G2位点甲基化水平与血压成负相关(Cp G2 and SBP:rs=-0.546,adjusted P=2.60×10-16;Cp G2 and DBP:rs=-0.539,adjusted P=7.60×10-16)。5、受试者工作特征曲线(Receiver operating characteristic curve,简称ROC曲线)分析:(1)TLR2基因:分析发现TLR2启动子Cp G1位点和Cp G6具有诊断价值(Cp G1:AUC(area under curve)=0.612,P=0.007;Cp G6:AUC(area under curve)=0.834,P=1.31×10-15);(2)IL6基因:分析发现IL6启动子Cp G2-3位点具有诊断学价值(Cp G2:AUC(area under curve)=0.638,P=0.001;Cp G3:AUC(area under curve)=0.704,P=8.12×10-7);(3)IFN-γ基因:分析发现IFN-γ启动子Cp G2位点具有诊断学价值(Cp G2:AUC(area under curve)=0.834,P=1.40×10-15)。结论:1、TLR2启动子Cp G6位点,IL6启动子Cp G3位点和IFN-γ启动子Cp G2位点的低甲基化是原发性高血压的危险因素。2、女性TLR2启动子Cp G6位点甲基化水平偏高,女性IL6启动子Cp G2位点和IFN-γ启动子Cp G1位点甲基化水平偏低。男性和女性基因甲基化水平存在差异。男性中,吸烟者和饮酒者的IL6启动子Cp G2-3甲基化水平降低,吸烟和饮酒作为EH的危险因素可能通过影响基因Cp G位点甲基化水平影响原发性高血压的发生发展。3、相关性分析发现TLR2启动子Cp G6位点、IL6启动子Cp G2-3位点甲基化水平与血压成负相关,IFN-γ启动子Cp G2位点甲基化水平与血压成负相关。4、TLR2启动子Cp G1和Cp G6位点,IL6启动子Cp G2-3位点和IFN-γ启动子Cp G2位点具有诊断价值。
[Abstract]:Objective: 1. To investigate the difference of DNA methylation of inflammatory factor gene TLR2, IL6 and IFN-gamma promoters between essential hypertension patients and normal controls; 2. To study the effect of gender, smoking and drinking on DNA methylation of inflammatory factor gene promoter Cp G site; 3. To explore the difference of DNA methylation of inflammatory factor gene promoter Cp G site on primary hypertension. Methods: 1. Using the method of multi-stage sampling, the subjects were included in the study. The demographic characteristics, environmental factors, physical examination and laboratory examination were conducted. Finally 96 new cases and 96 controls matched by sex and age 1:1 were determined. The baseline data of 192 subjects and blood samples were obtained. 2. DNA extraction, sulfite modification and pyrophosphate sequencing were used to detect the methylation level of TLR2, IL6 and IFN-gamma promoters in blood samples; 3. The methylation degree of inflammatory factor genes between the two groups was compared and the difference of methylation degree was analyzed. Results: 1. Comparison of the methylation levels of each gene: (1) TLR2 gene: The methylation level of TLR2 promoter Cp G6 in the new case group was lower than that in the normal control group (control group, VS new case group (%): (2) IL 6 gene: The methylation level of IL 6 promoter Cp G3 in the new case group was lower than that in the normal control group (control group: Cp G3: 57.45 [8.29VS] 51.52 [6.18], OR (95% CI) = 0.91 (0.861-0.979), adjusted P = 0.009); (2) IL 6 gene: The methylation level of IL 6 promoter Cp G3 in the new case group was lower than that in the normal control group (control group: Cp G3: 57.45 [8.29VS] 51.52 [6.18, OR (95% CI) = 0.90 (0.838-0.965), adjusted P = 0.004);(control group N-gamma gene: The methylation level of Cp G2 site of IFN-gamma promoter in the new case group was lower than that in the normal control group (control group vs new case group (%): Cp G2: 10.50 (%) 0.92VS 9.23 (%) 0.83, OR (95% CI) = 0.64 (0.480-0.856), adjusted P = 0.003). 2, sex comparison in the control group: (1) TLR2 gene: female TLR2 promoter Cp G6 site A (2) IL6 gene: female IL6 promoter Cp G2 site methylation level is low (male VS female (%): Cp G2: 65.53 [7.31] VS 60.26 [10.46], adjusted P = 0.046; (3) IFN - gamma gene: symbol rank and test analysis found that the female IFN - gamma promoter Cp G1 site Low methylation level (male vs female (%): Cp G1: 37.58 (%) vs 1.31 vs 36.52 (%) vs 1.64, adjusted P = 0.020). Correlation analysis between smoking, drinking and DNA methylation of inflammatory factor genes in male: (1) IL 6 gene: In male, the methylation level of IL 6 promoter Cp G2-3 in smokers decreased (non-smokers vs smokers (%): Cp G2: 64.28 (%) vs 6.36 vs 60.41 (%) vs 7.74, a Djusted P = 0.023; Cp G3: 57.78 [7.87 VS = 53.70 [(adjusted P = 0.038); CpG 2-3 methylation in IL 6 promoter Cp G2-3 decreased in drinkers (non-drinkers (%): Cp G2: 64.70 [7.03 VS 60.89 [.89] [7.89 [.32, adjusted P = 0.038; Cp G3: 0.03; Cp G3: 57.78 [: 60.48 [7.48 [7.58 VS 53.53.23 [.23 [53] [7.23 [7.99, adjusted P = adjusted P = 4.24-4); (2) differentiation site Point 4, Correlation analysis: (1) TLR2 gene: TLR2 promoter Cp G6 site methylation level was negatively correlated with blood pressure (Cp G6 and SBP: r = - 0.329, adjusted P = 3.95 *10-5; Cp G6 and DBP: r = - 0.304, adjusted P = 1.80 *10-4); IL6 gene: IL6 promoter Cp G2-3 site methylation level was negatively correlated with blood pressure (Cp G2 and SBP: r = - 0.274, adjusted P = 0.0.0). 09; Cp G2 and DBP: r = - 0.183, adjusted P = 0.011; Cp G3 and SBP: r = - 0.321, adjusted P = 5.60 x 10-6; Cp G3 and DBP: r = - 0.274, adjusted P: adjusted P: r = - 0.274, adjusted P = 1.17 x 10-4); (3) IFN-gamgene: IFN-gampromoter CpG2methylation level of CpG2promoter CpG2promoter CpG2promoter methylation level was negatively correlwith blood pressure (Cp G2 and SBP: rs = - 0.546, adjusted P = - 0.546, adjusted P = 2.60 x 10-16; Cp G3 and DBP: r = - 0.274: r = - 0 9, adjusted P = 7.60 5, Receiver operating characteristic curve (ROC curve) analysis: (1) TLR2 gene: analysis found that TLR2 promoter Cp G1 locus and Cp G6 have diagnostic value (Cp G1: AUC (area under curve) = 0.612, P = 0.007; Cp G6: AUC (area under curve) = 0.834, P = 1.31 *10-15); (2) IL6 gene: analysis found that IL6: IL6 gene Promoter Cp G2-3 locus has diagnostic value (Cp G2: AUC (area under curve) = 0.638, P = 0.001; Cp G3: AUC (area under curve) = 0.704, P = 8.12 *10-7); (3) IFN-gamma gene: analysis found that IFN-gamma promoter Cp G2 locus has diagnostic value (Cp G2: AUC (area under curve) = 0.834, P = 1.40 *10-15). Hypomethylation at Cp G3 and Cp G2 loci of IFN-gamma promoters is a risk factor for essential hypertension. 2. High methylation at Cp G6 locus in female TLR2 promoter, low methylation at Cp G2 locus and Cp G1 locus in female IL6 promoter. Differences in gene methylation levels between men and women. Smoking in men The methylation level of IL 6 promoter Cp G2-3 was decreased in both subjects and drinkers. Smoking and drinking as risk factors for EH may affect the occurrence and development of essential hypertension by affecting the methylation level of Cp G locus. 3. Correlation analysis showed that the methylation level of TLR2 promoter Cp G6 locus and IL 6 promoter Cp G2-3 locus was negatively correlated with blood pressure. The methylation level at Cp G2 site of - gamma promoter was negatively correlated with blood pressure. 4. TLR2 promoter Cp G1 and Cp G6 sites, IL6 promoter Cp G2-3 sites and IFN-gamma promoter Cp G2 sites were of diagnostic value.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R544.11
本文编号：2238259
[Abstract]:Objective: 1. To investigate the difference of DNA methylation of inflammatory factor gene TLR2, IL6 and IFN-gamma promoters between essential hypertension patients and normal controls; 2. To study the effect of gender, smoking and drinking on DNA methylation of inflammatory factor gene promoter Cp G site; 3. To explore the difference of DNA methylation of inflammatory factor gene promoter Cp G site on primary hypertension. Methods: 1. Using the method of multi-stage sampling, the subjects were included in the study. The demographic characteristics, environmental factors, physical examination and laboratory examination were conducted. Finally 96 new cases and 96 controls matched by sex and age 1:1 were determined. The baseline data of 192 subjects and blood samples were obtained. 2. DNA extraction, sulfite modification and pyrophosphate sequencing were used to detect the methylation level of TLR2, IL6 and IFN-gamma promoters in blood samples; 3. The methylation degree of inflammatory factor genes between the two groups was compared and the difference of methylation degree was analyzed. Results: 1. Comparison of the methylation levels of each gene: (1) TLR2 gene: The methylation level of TLR2 promoter Cp G6 in the new case group was lower than that in the normal control group (control group, VS new case group (%): (2) IL 6 gene: The methylation level of IL 6 promoter Cp G3 in the new case group was lower than that in the normal control group (control group: Cp G3: 57.45 [8.29VS] 51.52 [6.18], OR (95% CI) = 0.91 (0.861-0.979), adjusted P = 0.009); (2) IL 6 gene: The methylation level of IL 6 promoter Cp G3 in the new case group was lower than that in the normal control group (control group: Cp G3: 57.45 [8.29VS] 51.52 [6.18, OR (95% CI) = 0.90 (0.838-0.965), adjusted P = 0.004);(control group N-gamma gene: The methylation level of Cp G2 site of IFN-gamma promoter in the new case group was lower than that in the normal control group (control group vs new case group (%): Cp G2: 10.50 (%) 0.92VS 9.23 (%) 0.83, OR (95% CI) = 0.64 (0.480-0.856), adjusted P = 0.003). 2, sex comparison in the control group: (1) TLR2 gene: female TLR2 promoter Cp G6 site A (2) IL6 gene: female IL6 promoter Cp G2 site methylation level is low (male VS female (%): Cp G2: 65.53 [7.31] VS 60.26 [10.46], adjusted P = 0.046; (3) IFN - gamma gene: symbol rank and test analysis found that the female IFN - gamma promoter Cp G1 site Low methylation level (male vs female (%): Cp G1: 37.58 (%) vs 1.31 vs 36.52 (%) vs 1.64, adjusted P = 0.020). Correlation analysis between smoking, drinking and DNA methylation of inflammatory factor genes in male: (1) IL 6 gene: In male, the methylation level of IL 6 promoter Cp G2-3 in smokers decreased (non-smokers vs smokers (%): Cp G2: 64.28 (%) vs 6.36 vs 60.41 (%) vs 7.74, a Djusted P = 0.023; Cp G3: 57.78 [7.87 VS = 53.70 [(adjusted P = 0.038); CpG 2-3 methylation in IL 6 promoter Cp G2-3 decreased in drinkers (non-drinkers (%): Cp G2: 64.70 [7.03 VS 60.89 [.89] [7.89 [.32, adjusted P = 0.038; Cp G3: 0.03; Cp G3: 57.78 [: 60.48 [7.48 [7.58 VS 53.53.23 [.23 [53] [7.23 [7.99, adjusted P = adjusted P = 4.24-4); (2) differentiation site Point 4, Correlation analysis: (1) TLR2 gene: TLR2 promoter Cp G6 site methylation level was negatively correlated with blood pressure (Cp G6 and SBP: r = - 0.329, adjusted P = 3.95 *10-5; Cp G6 and DBP: r = - 0.304, adjusted P = 1.80 *10-4); IL6 gene: IL6 promoter Cp G2-3 site methylation level was negatively correlated with blood pressure (Cp G2 and SBP: r = - 0.274, adjusted P = 0.0.0). 09; Cp G2 and DBP: r = - 0.183, adjusted P = 0.011; Cp G3 and SBP: r = - 0.321, adjusted P = 5.60 x 10-6; Cp G3 and DBP: r = - 0.274, adjusted P: adjusted P: r = - 0.274, adjusted P = 1.17 x 10-4); (3) IFN-gamgene: IFN-gampromoter CpG2methylation level of CpG2promoter CpG2promoter CpG2promoter methylation level was negatively correlwith blood pressure (Cp G2 and SBP: rs = - 0.546, adjusted P = - 0.546, adjusted P = 2.60 x 10-16; Cp G3 and DBP: r = - 0.274: r = - 0 9, adjusted P = 7.60 5, Receiver operating characteristic curve (ROC curve) analysis: (1) TLR2 gene: analysis found that TLR2 promoter Cp G1 locus and Cp G6 have diagnostic value (Cp G1: AUC (area under curve) = 0.612, P = 0.007; Cp G6: AUC (area under curve) = 0.834, P = 1.31 *10-15); (2) IL6 gene: analysis found that IL6: IL6 gene Promoter Cp G2-3 locus has diagnostic value (Cp G2: AUC (area under curve) = 0.638, P = 0.001; Cp G3: AUC (area under curve) = 0.704, P = 8.12 *10-7); (3) IFN-gamma gene: analysis found that IFN-gamma promoter Cp G2 locus has diagnostic value (Cp G2: AUC (area under curve) = 0.834, P = 1.40 *10-15). Hypomethylation at Cp G3 and Cp G2 loci of IFN-gamma promoters is a risk factor for essential hypertension. 2. High methylation at Cp G6 locus in female TLR2 promoter, low methylation at Cp G2 locus and Cp G1 locus in female IL6 promoter. Differences in gene methylation levels between men and women. Smoking in men The methylation level of IL 6 promoter Cp G2-3 was decreased in both subjects and drinkers. Smoking and drinking as risk factors for EH may affect the occurrence and development of essential hypertension by affecting the methylation level of Cp G locus. 3. Correlation analysis showed that the methylation level of TLR2 promoter Cp G6 locus and IL 6 promoter Cp G2-3 locus was negatively correlated with blood pressure. The methylation level at Cp G2 site of - gamma promoter was negatively correlated with blood pressure. 4. TLR2 promoter Cp G1 and Cp G6 sites, IL6 promoter Cp G2-3 sites and IFN-gamma promoter Cp G2 sites were of diagnostic value.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R544.11
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