本文選題：原發(fā)性高血壓 + DNA甲基化　； 參考：《福建醫(yī)科大學》2016年博士論文

【摘要】：研究背景原發(fā)性高血壓(essential hypertension,EH)是一種原因不明,以動脈血壓升高為主要臨床表現(xiàn)的心血管綜合征,具有高度遺傳的特點。雖然全基因組關聯(lián)性研究(genome-wide association studies,GWAS)已經(jīng)識別了許多與血壓升高相關的基因位點,但在一般人群中這些基因位點的聯(lián)合效應只能解釋一小部分的血壓可遺傳性,EH這種“遺傳性缺失”的原因目前尚不清楚。關于“程序化高血壓”的動物研究表明某些基因的DNA甲基化表觀遺傳修飾可介導生命早期不良環(huán)境因素所致的成年高血壓。自發(fā)性高血壓大鼠(spontaneously hypertensive rats,SHR)是人類EH的理想動物模型,現(xiàn)發(fā)現(xiàn)其成年期的血壓水平也受出生前后的環(huán)境因素影響,由此推測特定基因的DNA甲基化參與EH病理過程的“程序化”。本團隊前期研究觀察到生命早期給予血管緊張素受體拮抗劑(angiotensin receptor blocker,ARB)氯沙坦(losartan,Los)能長期延緩SHR血壓的升高,抑制左室肥厚(left ventricular hypertrophy,LVH),持續(xù)下調(diào)腎素血管緊張素系統(tǒng)(renin-angiotensin system)的關鍵基因—血管緊張素1型受體(angiotensin type 1 receptor,AT1R)的表達。血管緊張素1型受體相關蛋白(angiotensin type 1 receptor-associated protein,ATRAP)是近年來發(fā)現(xiàn)的RAS新組分,它可負向調(diào)控AT1R信號通路。過表達ATRAP可抑制LVH的發(fā)展,ARB可上調(diào)ATRAP的表達。本課題將研究氯沙坦早期干預長期抑制SHR LVH過程中ATRAP基因甲基化的作用。力圖從表觀遺傳學角度闡述EH的“遺傳性缺失”現(xiàn)象,也為實現(xiàn)短期干預、長期降壓的治療理念進行初步的嘗試。目的1.探討Los早期干預對SHR的血壓、LVH以及心肌中ATRAP表達的長期影響。2.觀察成年shr與正常血壓的wista-kyoto(wky)大鼠心肌中atrap基因啟動子區(qū)dna甲基化的差異,探討los早期干預對atrap基因dna甲基化的長期影響。3.觀察los對血管緊張素ii(angiotensinii,angii)誘導的h9c2心肌細胞蛋白合成和atrap表達的影響。研究dna甲基化在los調(diào)控angii誘導的atrap表達及蛋白合成的作用。方法1動物實驗(1)實驗設計:將妊娠的shr母鼠分為四組:shr未干預(untreatedshr,shrun)組、shr生前干預(prenataltreatedshr,shrpre)組、shr生后干預(postnataltreatedshr,shrpost)組、shr序貫干預(sequentialtreatedshr,shrseq)組。將wky未干預(untreatedwky,wkyun)組作為對照組。shrpre、shrpost和shrseq都看作los早期干預組。wkyun和shrun的母鼠及子鼠均不接受los干預。shrpre指在孕期,母鼠接受los灌胃。shrpost指從哺乳期開始,母鼠接受los灌胃;斷乳后,子鼠繼續(xù)接受los灌胃至8周齡。shrseq在孕期、哺乳期,母鼠均接受氯沙坦灌胃;斷乳后,子鼠繼續(xù)接受los灌胃至8周齡。los干預劑量為30mg/kg/d。子鼠8周齡后,停止干預,繼續(xù)觀察8周,將16周齡的雄性子鼠作為實驗對象,每組子鼠8只。(2)血壓、左室形態(tài)及功能、atrap基因表達的研究:尾測法測量子鼠的收縮壓。心導管法測定大鼠左室血液動力學,收集左室收縮功能的指標:左室壓力最大上升速率(maximalrateofincreaseofleftventriclepressuredevelopment,lv+dp/dtmax)、左室收縮末壓(leftventricleendsystolicpressure,lvesp)和左室舒張功能的指標:左室壓力最大下降速率(maximalrateofdecreaseofleftventriclepressuredevelopment,lv-dp/dtmax)、左室舒張末壓(leftventricleenddiastolicpressure,lvedp)、等容舒張期壓力下降的時間常數(shù)(timeconstantofleftventricularisovolumerelaxation,t)。稱重法測定體質(zhì)量(bodyweight,bw)、左室質(zhì)量(leftventriclemass,lvm)并計算左室質(zhì)量/體質(zhì)量(leftventriclemass/bodyweight,lvm/bw)。天狼星紅染色后應用imageproplus4.5圖像分析系統(tǒng)測量左室心肌膠原容積分數(shù)(collagenvolumefraction,cvf)。elisa方法測定血漿和心肌中angii、i型膠原(collagenigel,coli)和iii型膠原(collageniiigel,coliii)的含量。real-timertpcr及westernblot檢測心肌中atrap的mrna及蛋白表達。(3)dna甲基化的研究:網(wǎng)站預測atrap基因啟動子區(qū)的cpg島并設計甲基化檢測引物。重亞硫酸氫鹽修飾后測序法(bisulfitesequencingpcr,bsp)測定atrap基因啟動子區(qū)轉(zhuǎn)錄起始位點上游9個cpg位點的甲基化水平。westernblot檢測dna甲基化轉(zhuǎn)移酶(dnamethyltransferase,dnmt)1、dnmt3a和dnmt3b的表達。染色質(zhì)免疫共沉淀(chromatinimmunoprecipitationassay,chipassay)分析三種dnmt與atrap基因的結(jié)合。2細胞實驗angii處理h9c2心肌細胞,首先觀察細胞蛋白合成速率、atrap表達、atrap基因啟動子區(qū)的甲基化水平、dnmt1、dnmt3a、dnmt3b表達以及與atrap基因的結(jié)合。接下來觀察los和dnmt抑制劑5-氮雜-2'-脫氧胞苷(5-aza-2'-deoxycytidine,5-aza)以及l(fā)os代謝產(chǎn)物5-羧酸洛沙坦(5-carboxylicacidlosartan,exp-3174)預處理后,angii所誘導的上述指標的變化。以3h亮氨酸摻入率反映心肌蛋白質(zhì)合成速率。3統(tǒng)計分析采用spss19.0軟件系統(tǒng)進行統(tǒng)計學分析。計量資料用(x±s)表示。動物實驗多組間比較采用單因素方差分析(one-wayanova),細胞實驗多組間比較采用one-wayanova或2x2析因分析,兩兩比較采用lsd(l)檢驗。以p0.05為差異有統(tǒng)計學意義。結(jié)果1.16周齡時,與shrun組相比,shrpre、shrpost和shrseq組的(1)血壓明顯降低[(189±20)、(174±16)、(173±17)比(212±22)mmhg,p0.05]。(2)lvm/bw顯著減少[(2.72±0.24)、(2.45±0.24)、(2.43±0.28)比(3.12±0.28)mg/g,p0.05]。(3)lv-dp/dtmax升高、t、lvedp降低。(4)心肌中cvf及coli、coliii型膠原含量明顯減少。(5)心肌中angii含量顯著降低,atrap的mrna和蛋白表達顯著升高(蛋白表達:0.10±0.02、0.12±0.02、0.13±0.04比0.04±0.01,p0.05)。2.atrap基因啟動子區(qū)有一個cpg島。與wkyun組相比,shrun組心肌中atrap基因轉(zhuǎn)錄起始位點上游4個cpg位點的甲基化水平升高。與shrun組相比,shrpre、SHRPost和SHRSeq組心肌中這4個位點的甲基化水平降低。5組大鼠心肌中DNMT1、DNMT3a和DNMT3b的表達差異無統(tǒng)計學意義。Chip assay顯示SHRUn組心肌中DNMT1、DNMT3a與ATRAP基因的結(jié)合較WKYUn組增強(DNMT1:2.9±0.3比1.0±0.0,DNMT3a:3.0±0.3比1.0±0.0,P0.05)。與SHRUn組相比,SHRPre、SHRPost和SHRSeq組心肌中DNMT1、DNMT3a與ATRAP基因的結(jié)合明顯減弱(DNMT1:0.9±0.1、0.9±0.1、1.0±0.1比2.9±0.3,DNMT3a:1.0±0.2、1.1±0.2、1.1±0.2比3.0±0.3,P0.05)。3.Ang II處理H9c2心肌細胞后,細胞蛋白合成速率明顯增加,ATRAPmRNA和蛋白表達下調(diào),ATRAP基因轉(zhuǎn)錄起始位點上游5個CpG位點的甲基化水平升高,DNMT1、DNMT3a表達上調(diào),與ATRAP基因的結(jié)合增強。Los、5-Aza或EXP-3174可不同程度地逆轉(zhuǎn)AngII所誘導的上述變化。結(jié)論1.Los早期干預能長期延緩成年SHR血壓的升高、抑制LVH和心肌纖維化,改善左室舒張功能,降低心肌中AngII含量,上調(diào)ATRAP表達。2.成年SHR心肌中ATRAP基因啟動子區(qū)的甲基化水平升高,DNMT1、DNMT3a與ATRAP基因的結(jié)合增強,Los早期干預能部分逆轉(zhuǎn)這種變化。3.Los能抑制AngII誘導H9c2心肌細胞的ATRAP表達下調(diào)及蛋白合成速率增加,這種作用與其降低DNMT1、DNMT3a所介導的ATRAP基因甲基化有關。4.Los早期干預長期抑制SHR LVH與ATRAP基因甲基化有關,提示表觀遺傳可能是EH“遺傳性缺失”的原因之一。
[Abstract]:Objective To study the effect of early intervention of losartan on the expression of AT1R in SHR , and to investigate the effect of early intervention on the expression of AT1R in SHR . After weaning , the rats received a los enema . After weaning , the rats received a los enema . After weaning , the rats received the los lavage . After weaning , the rats received the los lavage . After weaning , the rats received the los lavage . After weaning , the rats received the los lavage . After weaning , the rats received the los lavage . After weaning , the rats received los lavage . After weaning , the rats received the los lavage . After weaning , the rats received the los lavage . After weaning , the rats received the los lavage . After weaning , the rats received the los lavage . After weaning , the rats received the los lavage . After weaning , the rats were treated for 8 weeks , and the 16 - week - old male mice were treated as experimental subjects , and 8 rats in each group . ( 2 ) The study of blood pressure , left ventricular morphology and function , atrap gene expression : tail measurement method to measure systolic blood pressure in rats . The indexes of left ventricular systolic function were measured by cardiac catheterization : left ventricular pressure maximum ascending rate ( lv + dp / dtmax ) , left ventricular systolic pressure ( lvesp ) and left ventricular diastolic function : left ventricular pressure maximum descending rate ( lv - dp / dtmax ) , left ventricular diastolic pressure ( lvedp ) , and constant volume relaxation time constant ( t ) . mass ( bw ) , left ventricular mass ( lvm ) were measured and the left ventricular mass / mass ( lvm / bw ) was calculated . The expression of atrap mRNA and protein in myocardium were measured by using imageproplus 4.5 image analysis system . The expression of atrap mRNA and protein in myocardium were determined by enzyme - linked immunosorbent assay ( ELISA ) . Compared with wkyun group , the expression of DNMT3a and DNMT3b in myocardium of SHRUn group decreased significantly ( DNMT3a : 2.9 鹵 0.3 vs 1.0 鹵 0.0 , DNMT3a : 3.0 鹵 0.3 vs 1.0 鹵 0.0 , P0.05 ) . Compared with SHRUn group , the binding of DNMT3a to ATRAP gene significantly decreased ( DNMT3a : 0.9 鹵 0.1 , 0.9 鹵 0.1 , 1.0 鹵 0.2 , 1.1 鹵 0.2 , 1.1 鹵 0.2 vs 3.0 鹵 0.3 , P0.05 ) .
【學位授予單位】：福建醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R544.1
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本文編號：2069838