EPHX2 rs751141基因多态性与糖尿病血管并发症相关性研究及其机制探讨

发布时间：2018-03-03 17:44

本文选题：糖尿病相关肾病　切入点：缺血性脑卒中　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:多项流行病学调查显示,2型糖尿病(diabetes mellitus,DM)是一种复杂的代谢紊乱疾病,长期高血糖会导致机体多器官受累并出现较严重的并发症。目前认为DM血管并发症中糖尿病相关肾病(diabetic kidney disease,DKD)以及缺血性脑卒中的发生与遗传等因素密切相关。近年来,可溶性环氧化物水解酶(soluble epoxide hydrolase,sEH)在肾脏疾病尤其是DKD以及缺血性脑卒中的发病中所起的作用逐渐被重视,sEH抑制剂的研究以及该基因敲除有可能成为DKD及缺血性脑卒中治疗的新靶点。编码sEH的EPHX2基因rs751141多态性(R287Q)能影响sEH酶活性并且与多种临床疾病有关,其与DKD及糖尿病缺血性脑卒中的相关性目前尚有待于进一步研究。研究目的:1.探讨EPHX2 rs751141基因多态性中A等位基因是否是DM血管并发症DKD和缺血性脑卒中的保护因素。2.研究EPHX2 rs751141基因多态性A等位基因与sEH水解酶活性的关系及其参与DKD的分子机制。研究方法:1.EPHX2 rs751141基因多态性与2型DM血管并发症相关性研究:1)EPHX2 rs751141基因多态性与DKD的相关性研究:870例2型DM患者均为中日友好医院2015年2月至2016年6月住院患者,其中DKD患者406例(男性258例、女性148例),对照组糖尿病不伴相关肾病(Non-diabetic kidney disease,Non-DKD)患者464例(男性271例、女性193例)。提取患者外周血DNA并用荧光探针法进行SNP检测。采用卡方检验方法分析基因型频率、等位基因频率以及Hardy-Weinberg平衡。统计学效力分析采用Quanto software version 1.2.4软件,所有数据采用SPSS17.0统计软件进行处理,当p0.05,认为有统计学差异。2)EPHX2 rs751141基因多态性与DM伴缺血性脑卒中相关性研究:626例2型DM患者均为中日友好医院2015年2月至2016年6月住院患者。其中DM伴缺血性脑卒中患者236例(男性127例、女性109例),对照组DM不伴缺血性脑卒中患者390例(男性241例、女性149例)。检测方法、数据处理及统计分析同1)。2.EPHX2多态性与sEH水解酶活性的关系及其参与DKD分子机制研究:1)不同突变类型sEH重组质粒构建及其与sEH水解酶活性的关系:以人的cDNA为模板,根据GeneBank中人EPHX2 mRNA CDS序列设计引物,利用p-UCT DNA连接试剂盒及点突变技术在野生型基础上诱导点突变,共构建4种单点突变和3种双点突变重组质粒,并将其亚克隆至真核表达载体pcDNA3.1/V5-His-A。以Lipofectamine2000转染试剂将8种不同类型的sEH重组质粒:EPHX2/pcDNA3.1/V5-His(WT)EPHX2/R287Q/pcDNA3.1/V5-His(R287Q)、EPHX2/R287Q/R103C/pcDNA3.1/V5-His(R287Q+R103C)、EPHX2/C154T/pcDNA3.1/V5-His(C154T)、PHX2/K55R/pcDNA3.1/V5-His(K55R)、EPHX2/R103C/pcDNA3.1/V5-His(R103C)、EPHX2/K55R/R103C/pcDNA3.1/V5-His(K55R+R103C)、EPHX2/K55R/C154T/pcDNA3.1/V5-His(K55R+C 154T)及pcDNA3.1/V5-His-A空载体(negtive control,NC)转染至HK2细胞,同时转染绿色荧光蛋白(green fluorescent protein,GFP)标签的重组质粒,荧光显微镜下观察转染效率,Western Blot检测融合蛋白的表达。将上述重组质粒转染HK2细胞,并将其分为两组,其中一组细胞培养液中补充足量的11,12-EET,另外一组在补充11,12-EET的同时加入sEH抑制剂TPPU。ELISA方法检测各组细胞培养液中的11,12-DHET含量,采用t检验比较不同类型sEH重组质粒的水解活性是否存在差异以及TPPU对水解酶活性的影响。2)R287Q突变型重组质粒参与DKD分子机制研究:将成功构建的WT组、R287Q突变型组和NC组重组质粒分别转染至HK2细胞,各分为两组,其中一组转染48h后收获细胞培养上清、RNA和蛋白,另外一组转染24h后给予11,12-EET共同培养后收获细胞培养上清、RNA和蛋白,检测NO、IL-17A以及IL-1等炎症因子水平、RT-PCR 检测 eNOS、IL-17A、IL-6 和 IL-1β 等 mRNA 表达水平及 Western blot 方法检测PI3K/AKT信号通路蛋白表达。研究结果:1.EPHX2 rs751141基因多态性与2型DM血管并发症相关性研究:1)EPHX2 rs751141基因多态性与DKD相关性研究结果:Non-DKD组rs751141 A等位基因频率明显高于DKD组(p0.01)。校正DKD相关风险因素后,在加性和隐性遗传模型下A等位基因携带者具有较低的DKD患病风险(OR=0.68,95%CI=0.52-0.88,p0.01;OR=0.28,95%CI=0.14-0.60,p0.01)。相关结果表明在高同型半胱氨酸时,在校正DKD相关风险因素后,三种遗传模型A等位基因携带者均具有比较低的DKD患病风险(p0.01,p0.05和p0.05)。2)EPHX2rs751141基因多态性与DM缺血性脑卒中相关性研究结果:DM不伴缺血性脑卒中组EPHX2 rs751141 A等位基因频率明显高于DM伴缺血性脑卒中组(p0.05)。校正相关风险因素后,在加性和显性遗传模型下,A等位基因携带者具有较低的缺血性脑卒中患病风险(OR=0.72,95%CI=0.55-0.97,p0.05;OR=0.67,95%CI=0.48-0.95,p0.05)。2.EPHX2rs751141多态性A等位基因与sEH水解酶活性的关系及其参与DKD的分子机制研究:1)不同类型sEH重组质粒与sEH水解酶活性的关系:成功构建了 1种野生型WT和 7 种突变型重组质粒(R287Q、R287Q+R103C、C154T、K55R、R103C、K55R+R103C、K55R+C154T),并建立HK-2高表达系统。其中,R287Q重组质粒组11,12-DHET水平明显低于WT组(p0.01);C154T重组质粒组明显高于WT组(p0.05)。细胞培养液中加入11,12-EET的同时给予TPPU,可显著下调11,12-DHET含量至基础水平。2)炎症因子及信号通路检测:突变型R287Q组细胞上清NO水平、IL-17A水平以及IL-1β水平明显低于WT组(p0.05)。相对于单纯WT组,加入11,12-EET共同培养的WT组炎症因子水平降低,但仍高于加入11,12-EET突变型R287Q组。RT-PCR结果也发现R287Q组中在eNOS、IL-17A、IL-6和IL-1β等的mRNA表达水平低于WT组,相对于单纯WT组,加入11,12-EET共同培养的WT组炎症因子mRNA表达水平降低,但仍高于加入11,12-EET突变型R287Q组。采用Western blot方法检测NC组、WT组和R287Q组蛋白发现R287Q组可以通过调控PI3K/AKT信号通路减轻DKD炎症。研究结论:1.EPHX2rs751141多态性中A等位基因对DM血管并发症DKD和缺血性脑卒中的发生均具有保护作用,提示该位点可能参与了 DM血管并发症的发生发展。2.在体外细胞实验中,突变型R287Q重组质粒相对于野生型WT具有较低的11,12-EET水解能力。突变型R287Q相对于野生型WT具有较低的炎症水平,外源性11,12-EET可以降低WT组炎症水平。A等位基因保护作用可能通过调控PI3K/AKT信号通路参与DKD的分子机制。
[Abstract]:Background: epidemiological surveys show a number of type 2 diabetes mellitus (diabetes, mellitus, DM) is a complex metabolic disorder, chronic hyperglycemia can cause the body organs and the emergence of more serious complications. The diabetic vascular complications associated with renal disease DM (diabetic kidney disease, DKD) and ischemic stroke the occurrence and genetic factors are closely related. In recent years, soluble epoxide hydrolase (soluble epoxide, hydrolase, sEH) in the pathogenesis of renal diseases especially DKD and ischemic stroke in the role is gradually paid more attention to the study of sEH inhibitors, and the knockout may become a new target for the treatment of ischemic stroke and DKD EPHX2. Rs751141 gene polymorphism encoding sEH (R287Q) can affect the activity of sEH and related to many clinical diseases, and DKD diabetes and ischemic stroke The association needs to be further studied. The research objective: 1. to investigate the EPHX2 polymorphism of rs751141 gene A allele is a protective factor for DM, DKD and vascular complications in ischemic stroke.2. study of EPHX2 rs751141 gene polymorphism of A alleles and sEH hydrolase activity and its molecular mechanism in DKD. Methods: Study on the correlation between 1.EPHX2 rs751141 gene polymorphism and vascular complications of type 2 DM: 1) study on the correlation between EPHX2 rs751141 gene polymorphism and DKD: 870 cases of type 2 DM patients were hospitalized patients in China-Japan Friendship Hospital from February 2015 to June 2016, including 406 cases of DKD patients (258 males, 148 females), the control group without diabetes nephropathy (Non-diabetic kidney disease, Non-DKD) in 464 patients (271 males, 193 females). Extracted peripheral blood DNA and SNP were detected by fluorescence probe method. By using the chi square test Method for analysis of genotype frequency and allele frequency and Hardy-Weinberg balance. Statistical validity analysis using the Quanto software version 1.2.4 software, all data using SPSS17.0 statistical software for processing, when P0.05, that there is a significant difference.2) correlation between ischemic stroke and EPHX2 polymorphisms of rs751141 gene and DM with 626 cases of type 2 DM patients China-Japan Friendship Hospital from February 2015 to June 2016. The patients with DM in patients with ischemic stroke in 236 cases (male 127 cases, female 109 cases), the control group of DM patients without ischemic stroke in 390 cases (241 males, 149 females). The detection method, data processing and statistical analysis with 1) the relationship between.2.EPHX2 polymorphism and sEH hydrolase activity and molecular mechanism of DKD involved in the study: 1) different mutant hydrolase activity and its relationship with sEH type sEH: the recombinant plasmid cDNA as template, root According to the GeneBank EPHX2 mRNA CDS sequence primers were designed using p-UCT DNA connection kit and a point mutation induced point mutations in the wild type on the basis of a total construction of 4 single point mutations and 3 double mutation recombinant plasmid and subcloned into eukaryotic expression vector pcDNA3.1/V5-His-A. by Lipofectamine2000 transfection reagent 8 different types of sEH recombinant plasmid EPHX2/pcDNA3.1/V5-His (WT) EPHX2/R287Q/pcDNA3.1/V5-His (R287Q), EPHX2/R287Q/R103C/pcDNA3.1/V5-His (R287Q+R103C), EPHX2/C154T/pcDNA3.1/V5-His (C154T), PHX2/K55R/pcDNA3.1/ V5-His (K55R), EPHX2/R103C/pcDNA3.1/V5-His (R103C), EPHX2/K55R/R103C/pcDNA3.1/V5-His (K55R+R103C), EPHX2/K55R/C154T/pcDNA3.1/V5-His (K55R+C 154T) and pcDNA3.1/V5-His-A (negtive control, NC empty vector) was transfected into HK2 cells, and transfection green fluorescent protein (green fluorescent, protein, GFP) The recombinant plasmid tag, the transfection efficiency was observed under fluorescence microscope to detect the expression of Western fusion protein Blot. The recombinant plasmid was transfected into HK2 cells, and it can be divided into two groups, one group of cell culture 11,12-EET added plenty of liquid, the other group added 11,12-EET and sEH inhibitor TPPU.ELISA was used to detect the cell the content of 11,12-DHET in culture medium, using t test comparison of type sEH hydrolysis activity of different recombinant plasmids and whether there are differences between the effects of TPPU on the enzymatic activity of R287Q mutant recombinant plasmid.2) participated in the study of molecular mechanism of DKD: the successful construction of WT group, R287Q group and NC group of mutant recombinant plasmids were transfected into HK2 cells each, divided into two groups, a group of cell culture supernatants harvested after transfection of 48h, RNA and protein, another group after transfection of 24h 11,12-EET cultured cells cultured on harvest Clearly, RNA and protein detection, NO, IL-17A, IL-1 and other inflammatory cytokines, RT-PCR detection of eNOS, IL-17A, expression of PI3K/AKT signaling pathway protein IL-6 and IL-1 beta mRNA expression level and Western blot method. Results: the correlation between 1.EPHX2 rs751141 gene polymorphism and vascular complications of type 2 DM: 1) EPHX2 rs751141 gene Study on the correlation between polymorphism and DKD results: Non-DKD group rs751141 A allele frequency was significantly higher than that of DKD group (P0.01). The correction DKD related risk factors, the additive and recessive genetic model A allele has a lower risk of DKD (OR=0.68,95%CI=0.52-0.88, P0.01; OR=0.28,95%CI=0.14-0.60, P0.01). The results show that in high homocysteine, in the correction of DKD related risk factors, three kinds of genetic models of A allele carriers had relatively low risk of DKD (P0.01, P0.05 and p0.0 5).2) EPHX2rs751141 gene polymorphism and ischemic stroke DM correlation research results: DM without ischemic stroke EPHX2 rs751141 A allele frequency was significantly higher than that of DM with cerebral ischemic stroke (P0.05). The related risk factors after correction, the additive and dominant genetic model, A allele carriers with ischemic stroke lower risk (OR=0.72,95%CI=0.55-0.97, P0.05; OR=0.67,95%CI=0.48-0.95, P0.05) on the molecular mechanism of the relationship between the.2.EPHX2rs751141 polymorphism of A alleles and sEH hydrolase activity in DKD: 1) and the relationship between different types of recombinant plasmid sEH and sEH hydrolase activity: successfully constructed 1 kinds of wild type WT and 7 the mutant recombinant plasmids (R287Q, R287Q+R103C, C154T, K55R, R103C, K55R+R103C, K55R+C154T), and establish a high expression level of 11,12-DHET HK-2. The recombinant plasmid of R287Q group was significantly lower than that of WT Group (P0.01); the recombinant plasmid of C154T group was significantly higher than WT group (P0.05). Cell culture medium added 11,12-EET TPPU, could significantly decrease the content of 11,12-DHET to basic level.2) inflammatory cytokines and signaling pathways: detection of mutant R287Q cells supernatant NO level, IL-17A level and IL-1 levels were lower than those of group WT (P0.05). Compared with WT group, WT group, inflammatory cytokines in cultured with 11,12-EET decreased, but still higher than the accession to the 11,12-EET mutant R287Q group.RT-PCR results were also found in the R287Q group at eNOS, IL-17A, IL-6 and IL-1 P mRNA expression level was lower than that of WT group compared with WT group, the expression level of WT group the inflammatory factors of mRNA cultured with 11,12-EET decreased, but still higher than the accession to the 11,12-EET mutant R287Q group. NC group were measured using Western blot method, WT group and R287Q group group R287Q protein through regulation of PI3K/AKT signaling The way to reduce DKD inflammation. Conclusion: the 1.EPHX2rs751141 polymorphism of A allele of DM DKD and vascular complications of ischemic stroke have protective effects, suggesting that the site may be involved in the occurrence and development of.2. DM and vascular complications in vitro, the recombinant plasmid of R287Q mutant compared with wild type WT has 11,12-EET hydrolysis ability low. R287Q mutant compared with wild type WT have lower levels of inflammation, exogenous 11,12-EET can reduce the level of inflammation in WT group.A allele by the possible protective effect of molecular mechanism of PI3K/AKT signaling pathway is involved in DKD.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2

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