本文選題：NADPH氧化酶　切入點：基因多態(tài)性　出處：《河北醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:糖尿病腎臟病(Diabetic kidney disease,DKD)是糖尿病最常見的微血管并發(fā)癥。DKD的發(fā)病機(jī)制與氧化應(yīng)激、炎癥反應(yīng)、易感基因及腎血流動力學(xué)改變等多方面因素有關(guān)。Brown Lee[1]提出糖尿病及其并發(fā)癥的統(tǒng)一機(jī)制學(xué)說,其核心是高糖引發(fā)的氧化應(yīng)激(Oxidative stress,OS)。NADPH氧化酶是催化氧自由基(Reactive oxygen species,ROS)生成的關(guān)鍵酶,廣泛存在于腎血管內(nèi)皮細(xì)胞、腎小球系膜細(xì)胞和足細(xì)胞,分子生物學(xué)研究發(fā)現(xiàn),NADPH氧化酶亞基p47phox第338位上存在G→A突變,這與ROS產(chǎn)生增加及DKD發(fā)生、發(fā)展密切相關(guān)[2]。8-羥基脫氧鳥苷(8-Hydroxy-2-deoxyguanosine,8-OHd G)是DNA氧化損傷的特異指標(biāo),因其影響因素少,在體內(nèi)含量穩(wěn)定,被公認(rèn)為評價機(jī)體氧化損傷程度的理想標(biāo)志物[3]。本研究的目的是①通過酶聯(lián)免疫吸附法(Enzyme-linked immunosorbent assay,ELISA)檢測2型糖尿病(Type 2 diabetes mellitus,T2DM)患者血清中8-OHd G含量,從而明確氧化應(yīng)激在糖尿病及其腎臟疾病發(fā)生中所起的作用;②通過聚合酶鏈反應(yīng)(Polymerase chain reaction,PCR)聯(lián)合單核苷酸多態(tài)性(Single nucleotide polymorphism,SNP)直接測序法探討河北地區(qū)漢族人群NADPH氧化酶p47phox基因G338A多態(tài)性與DKD的發(fā)病及其病情程度關(guān)系,旨在對DKD的診斷及防治提供分子遺傳學(xué)的理論依據(jù)。方法:依據(jù)1999年WHO糖尿病診斷和分型標(biāo)準(zhǔn),選取本院內(nèi)分泌科住院的T2DM患者198例,根據(jù)尿白蛋白與肌酐比值(尿A/Cr)將患者分為三組,A:單純T2DM(Simple diabetes,SDM)組70例,其中男43例,女27例,年齡56.20±7.74歲,病程6.02±2.08年;B:微量白蛋白尿組65例,其中男41例,女24例,年齡58.09±9.42歲,病程9.73±4.59年;C:大量白蛋白尿組63例,其中男39例,女24例,年齡57.89±11.17歲,病程10.47±5.21年。正常對照(Normal control,NC)組為同期我院醫(yī)務(wù)人員及常規(guī)體檢者中健康個體70例,其中男42例,女28例,年齡55.60±10.81歲。入選者均為無親緣關(guān)系河北地區(qū)漢族人。正常對照組和試驗組性別與年齡匹配。本研究已通過倫理委員會審批,簽署知情同意原則后,抽取受試者(禁食12h后)肘靜脈血3ml,乙二胺乙酸(Ethylene diamine tetraacetic acid,EDTA)抗凝,用于提取基因組DNA;4ml靜脈血,分離血清,用于檢測各項生化指標(biāo)和8-OHd G。(1)臨床資料收集:包括性別、年齡、病程、身高(cm)、體重(kg)、血壓(mm Hg)等,計算體質(zhì)量指數(shù)(Body mass index,BMI)=體重/身高2;(2)血生化指標(biāo)檢查:使用全自動生化儀測定空腹血糖(Fasting plasma glucose,FBG)、總膽固醇(Total cholesterol,TC)、甘油三酯(Triglycerides,TG)、高密度脂蛋白膽固醇(High-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白膽固醇(Low-density lipoprotein cholesterol,LDL-C)及肝腎功能;(3)免疫比濁法測定糖化血紅蛋白(Hb A1c)及尿微量白蛋白、肌酐比值(尿A/Cr);(4)酶聯(lián)免疫吸附法(ELISA)測定血清8-OHd G水平。(5)p47phox G338A基因多態(tài)性的測定:①基因組DNA提取:3ml的2%乙二胺乙酸(EDTA)抗凝血,用血液基因組DNA小量提取試劑盒提取外周血白細(xì)胞DNA,然后經(jīng)紫光分光光度儀測DNA濃度及純度。②聚合酶鏈反應(yīng)(PCR)聯(lián)合單核苷酸多態(tài)性(SNP)直接測序法檢測p47phox G338A基因型:以外周血白細(xì)胞基因組DNA為模板進(jìn)行PCR擴(kuò)增,用于擴(kuò)增NADPH p47phox基因的引物序列根據(jù)基因文庫的目的擴(kuò)增區(qū),經(jīng)Primer Premier 5軟件設(shè)計,然后由上海捷瑞生物工程有限公司合成,引物序列和擴(kuò)增片段如下:名稱引物序列擴(kuò)增長度NADPHp47phoxF:5'-TCCCAAGTGGTTTGACGG-3 298bpR:5'-CCTCCTCTTTCTGGCTGTG-3 PCR總反應(yīng)體系25ul,其中含12.5ul PCR Premix,8.5ul滅菌雙蒸餾水,上下游引物各1ul,模板DNA 2ul。PCR反應(yīng)條件:94℃預(yù)變性3 min,94℃變性30 s,54℃退火30 s,72℃延伸40 s,完成30次循環(huán),末次循環(huán)后,在72℃充分延伸3 min,4℃冷卻10 min。③進(jìn)行PCR反應(yīng)產(chǎn)物的純化,PCR產(chǎn)物純化試劑盒由上海捷瑞生物工程有限公司提供。④純化后的PCR產(chǎn)物經(jīng)SNP測序儀進(jìn)行測序,測序結(jié)果在Genbank數(shù)據(jù)庫中進(jìn)行比較分析,證實P47phox基因第4外顯子G338A多態(tài)性有GG、GA、AA三種基因型。(6)統(tǒng)計學(xué)處理:應(yīng)用SPSS 16.0統(tǒng)計軟件對結(jié)果進(jìn)行統(tǒng)計學(xué)處理,對各組多態(tài)位點基因分布進(jìn)行Hardy-Weinberg平衡吻合度檢測;計量資料進(jìn)行正態(tài)性檢驗,統(tǒng)計描述以均值±標(biāo)準(zhǔn)差(X±s)表示;組間比較用方差分析;基因型頻率和等位基因頻率的比較用卡方檢驗;8-OHd G濃度與其他因素的相關(guān)性采用Spearman相關(guān)分析法;用多元逐步回歸法分析各因素在T2DM腎臟病變發(fā)生中各自獨立的作用及相對影響大小。結(jié)果:①正常對照組與T2DM組的年齡、性別構(gòu)成比無差異(P0.05),具有可比性;與正常對照組比較,T2DM組的BMI、SBP、DBP、FBG、TC、TG及LDL-C均明顯升高,HDL-C明顯降低(P0.05或P0.01);與單純T2DM組相比,DKD兩組的病程、SBP、DBP、TC及TG均顯著升高,HDL-C明顯降低(P0.05或P0.01);與微量白蛋白尿組相比,大量白蛋白尿組病程、SBP、DBP明顯升高,LDL-C則明顯降低(P0.05),HDL-C則無明顯差異(見Fig.1,Table 1)。②單純T2DM組血清8-OHd G水平(5.02±0.69)較正常對照組(3.03±0.64)明顯升高(P0.05);DKD兩組血清8-OHd G水平均明顯高于正常對照組和單純T2DM組(P0.01),且大量白蛋白尿組(9.33±1.71)較微量白蛋白尿組(7.44±0.97)顯著升高(P0.05)(見Fig.2)。③血清8-OHd G與病程(r=0.264,P=0.001)、SBP(r=0.195,P=0.004)、FBG(r=0.217,P=0.000)、Hb A1c(r=0.198,P=0.005)、TC(r=0.173,P=0.008)、TG(r=0.296,P=0.000)和LDL-C(r=0.187,P=0.007)呈顯著正相關(guān),與HDL-C呈顯著負(fù)相關(guān)(r=-0.271,P=0.000)(見Table 2)。④正常對照組、單純T2DM組、微量白蛋白尿組及大量白蛋白尿組GA+AA基因型頻率分別為11.4%、18.6%、32.3%和38.1%,A等位基因頻率分別為5.7%、10.0%、17.7%和21.4%。比較各組間的GA+AA基因型和A等位基因頻率,單純T2DM組與正常對照組間無顯著性差異;DKD兩組該頻率均明顯高于正常對照組和單純T2DM組;而DKD兩組間該頻率無顯著性差異(見Fig.6,Table 3)。⑤不同基因型組間年齡、性別、DBP、TC、TG、LDL-C、HDL-C均無顯著性差異(P0.05);與GG型相比,GA+AA型的病程、Hb A1c、FBG、SBP及8-OHd G均明顯升高(P0.05或P0.01)。GA+AA基因型組中DKD發(fā)病率為68.2%,顯著高于GG基因型組的41.1%(X2為14.635,P0.01);GA+AA基因型與GG基因型相比,DKD發(fā)生率的危險度OR=3.072,OR值的95%可信區(qū)間為(1.705,5.537),即GA+AA基因型患DKD的風(fēng)險約是GG基因型的3倍(見Fig.7、8,Table 4、5)。⑥多元逐步回歸分析顯示:病程(β=0.257,P=0.036)、LDL-C(β=0.357,P=0.015)、SBP(β=0.291,P=0.019)、FBG(β=0.285,P=0.022)和8-OHd G(β=0.473,P=0.008)是DKD的獨立危險因素,而A等位基因并不是DKD的獨立危險因素(見Table 6)。結(jié)論:1 T2DM患者體內(nèi)8-OHd G水平升高,提示T2DM患者體內(nèi)存在氧化應(yīng)激。2河北地區(qū)漢族T2DM人群存在p47phox G338A基因多態(tài)性,A等位基因攜帶者血清8-OHd G水平較高。3 8-OHd G是DKD發(fā)生的獨立危險因素,但A等位基因可能不是DKD的易感基因。
[Abstract]:Objective: diabetic kidney disease (Diabetic kidney, disease, DKD) is associated with the pathogenesis of oxidative stress,.DKD is the most common microvascular complications of diabetes inflammation, susceptibility gene and renal hemodynamic changes and other factors related to the.Brown Lee[1] of diabetes and its complications, unifying mechanism theory, its core is the oxidative stress caused by high glucose (Oxidative stress, OS).NADPH oxidase catalytic oxygen free radical (Reactive oxygen, species, ROS) key enzyme generated, widely exists in renal vascular endothelial cells, mesangial cells and podocytes, molecular biology research found that NADPH oxidase subunit p47phox 338th on G, A and ROS have increased the mutation. And DKD, [2].8- are closely related to the development of 8-hydroxy-2-deoxyguanosine (8-Hydroxy-2-deoxyguanosine 8-OHd G) is a specific marker of oxidative damage to DNA, because of its less influence factors, in vivo The content and stability, is recognized as an ideal marker for [3]. evaluation of oxidative damage the body the purpose of this study is through enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent, assay, ELISA) detection of type 2 diabetes (Type 2 diabetes mellitus 8-OHd G, T2DM) content in serum, so as to clear the oxidative stress in diabetes and disease the role of the kidney; by polymerase chain reaction (Polymerase chain reaction, PCR single nucleotide polymorphism (Single) combined with nucleotide polymorphism, SNP) to investigate the direct sequencing of Han population in Hebei area NADPH oxidase p47phox gene G338A polymorphism and the pathogenesis of DKD and its relation to severity of disease, diagnosis and prevention of DKD and provide a theoretical basis for molecular genetics. Methods: according to the 1999 WHO diabetes diagnosis and classification standard, a total of 198 T2DM patients in our hospital department of Endocrinology hospitalized cases, root 鎹翱鐧借泲鐧戒笌鑲岄厫姣斿，

本文編號：1617162