氯沙坦早期干预抑制自发性高血压大鼠左室肥厚过程中ATRAP基因甲基化的作用

发布时间：2018-06-26 08:54

  本文选题：原发性高血压 + DNA甲基化　； 参考：《福建医科大学》2016年博士论文

【摘要】：研究背景原发性高血压(essential hypertension,EH)是一种原因不明,以动脉血压升高为主要临床表现的心血管综合征,具有高度遗传的特点。虽然全基因组关联性研究(genome-wide association studies,GWAS)已经识别了许多与血压升高相关的基因位点,但在一般人群中这些基因位点的联合效应只能解释一小部分的血压可遗传性,EH这种“遗传性缺失”的原因目前尚不清楚。关于“程序化高血压”的动物研究表明某些基因的DNA甲基化表观遗传修饰可介导生命早期不良环境因素所致的成年高血压。自发性高血压大鼠(spontaneously hypertensive rats,SHR)是人类EH的理想动物模型,现发现其成年期的血压水平也受出生前后的环境因素影响,由此推测特定基因的DNA甲基化参与EH病理过程的“程序化”。本团队前期研究观察到生命早期给予血管紧张素受体拮抗剂(angiotensin receptor blocker,ARB)氯沙坦(losartan,Los)能长期延缓SHR血压的升高,抑制左室肥厚(left ventricular hypertrophy,LVH),持续下调肾素血管紧张素系统(renin-angiotensin system)的关键基因—血管紧张素1型受体(angiotensin type 1 receptor,AT1R)的表达。血管紧张素1型受体相关蛋白(angiotensin type 1 receptor-associated protein,ATRAP)是近年来发现的RAS新组分,它可负向调控AT1R信号通路。过表达ATRAP可抑制LVH的发展,ARB可上调ATRAP的表达。本课题将研究氯沙坦早期干预长期抑制SHR LVH过程中ATRAP基因甲基化的作用。力图从表观遗传学角度阐述EH的“遗传性缺失”现象,也为实现短期干预、长期降压的治疗理念进行初步的尝试。目的1.探讨Los早期干预对SHR的血压、LVH以及心肌中ATRAP表达的长期影响。2.观察成年shr与正常血压的wista-kyoto(wky)大鼠心肌中atrap基因启动子区dna甲基化的差异,探讨los早期干预对atrap基因dna甲基化的长期影响。3.观察los对血管紧张素ii(angiotensinii,angii)诱导的h9c2心肌细胞蛋白合成和atrap表达的影响。研究dna甲基化在los调控angii诱导的atrap表达及蛋白合成的作用。方法1动物实验(1)实验设计:将妊娠的shr母鼠分为四组:shr未干预(untreatedshr,shrun)组、shr生前干预(prenataltreatedshr,shrpre)组、shr生后干预(postnataltreatedshr,shrpost)组、shr序贯干预(sequentialtreatedshr,shrseq)组。将wky未干预(untreatedwky,wkyun)组作为对照组。shrpre、shrpost和shrseq都看作los早期干预组。wkyun和shrun的母鼠及子鼠均不接受los干预。shrpre指在孕期,母鼠接受los灌胃。shrpost指从哺乳期开始,母鼠接受los灌胃;断乳后,子鼠继续接受los灌胃至8周龄。shrseq在孕期、哺乳期,母鼠均接受氯沙坦灌胃;断乳后,子鼠继续接受los灌胃至8周龄。los干预剂量为30mg/kg/d。子鼠8周龄后,停止干预,继续观察8周,将16周龄的雄性子鼠作为实验对象,每组子鼠8只。(2)血压、左室形态及功能、atrap基因表达的研究:尾测法测量子鼠的收缩压。心导管法测定大鼠左室血液动力学,收集左室收缩功能的指标:左室压力最大上升速率(maximalrateofincreaseofleftventriclepressuredevelopment,lv+dp/dtmax)、左室收缩末压(leftventricleendsystolicpressure,lvesp)和左室舒张功能的指标:左室压力最大下降速率(maximalrateofdecreaseofleftventriclepressuredevelopment,lv-dp/dtmax)、左室舒张末压(leftventricleenddiastolicpressure,lvedp)、等容舒张期压力下降的时间常数(timeconstantofleftventricularisovolumerelaxation,t)。称重法测定体质量(bodyweight,bw)、左室质量(leftventriclemass,lvm)并计算左室质量/体质量(leftventriclemass/bodyweight,lvm/bw)。天狼星红染色后应用imageproplus4.5图像分析系统测量左室心肌胶原容积分数(collagenvolumefraction,cvf)。elisa方法测定血浆和心肌中angii、i型胶原(collagenigel,coli)和iii型胶原(collageniiigel,coliii)的含量。real-timertpcr及westernblot检测心肌中atrap的mrna及蛋白表达。(3)dna甲基化的研究:网站预测atrap基因启动子区的cpg岛并设计甲基化检测引物。重亚硫酸氢盐修饰后测序法(bisulfitesequencingpcr,bsp)测定atrap基因启动子区转录起始位点上游9个cpg位点的甲基化水平。westernblot检测dna甲基化转移酶(dnamethyltransferase,dnmt)1、dnmt3a和dnmt3b的表达。染色质免疫共沉淀(chromatinimmunoprecipitationassay,chipassay)分析三种dnmt与atrap基因的结合。2细胞实验angii处理h9c2心肌细胞,首先观察细胞蛋白合成速率、atrap表达、atrap基因启动子区的甲基化水平、dnmt1、dnmt3a、dnmt3b表达以及与atrap基因的结合。接下来观察los和dnmt抑制剂5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-aza)以及los代谢产物5-羧酸洛沙坦(5-carboxylicacidlosartan,exp-3174)预处理后,angii所诱导的上述指标的变化。以3h亮氨酸掺入率反映心肌蛋白质合成速率。3统计分析采用spss19.0软件系统进行统计学分析。计量资料用(x±s)表示。动物实验多组间比较采用单因素方差分析(one-wayanova),细胞实验多组间比较采用one-wayanova或2x2析因分析,两两比较采用lsd(l)检验。以p0.05为差异有统计学意义。结果1.16周龄时,与shrun组相比,shrpre、shrpost和shrseq组的(1)血压明显降低[(189±20)、(174±16)、(173±17)比(212±22)mmhg,p0.05]。(2)lvm/bw显著减少[(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型胶原含量明显减少。(5)心肌中angii含量显著降低,atrap的mrna和蛋白表达显著升高(蛋白表达:0.10±0.02、0.12±0.02、0.13±0.04比0.04±0.01,p0.05)。2.atrap基因启动子区有一个cpg岛。与wkyun组相比,shrun组心肌中atrap基因转录起始位点上游4个cpg位点的甲基化水平升高。与shrun组相比,shrpre、SHRPost和SHRSeq组心肌中这4个位点的甲基化水平降低。5组大鼠心肌中DNMT1、DNMT3a和DNMT3b的表达差异无统计学意义。Chip assay显示SHRUn组心肌中DNMT1、DNMT3a与ATRAP基因的结合较WKYUn组增强(DNMT1:2.9±0.3比1.0±0.0,DNMT3a:3.0±0.3比1.0±0.0,P0.05)。与SHRUn组相比,SHRPre、SHRPost和SHRSeq组心肌中DNMT1、DNMT3a与ATRAP基因的结合明显减弱(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处理H9c2心肌细胞后,细胞蛋白合成速率明显增加,ATRAPmRNA和蛋白表达下调,ATRAP基因转录起始位点上游5个CpG位点的甲基化水平升高,DNMT1、DNMT3a表达上调,与ATRAP基因的结合增强。Los、5-Aza或EXP-3174可不同程度地逆转AngII所诱导的上述变化。结论1.Los早期干预能长期延缓成年SHR血压的升高、抑制LVH和心肌纤维化,改善左室舒张功能,降低心肌中AngII含量,上调ATRAP表达。2.成年SHR心肌中ATRAP基因启动子区的甲基化水平升高,DNMT1、DNMT3a与ATRAP基因的结合增强,Los早期干预能部分逆转这种变化。3.Los能抑制AngII诱导H9c2心肌细胞的ATRAP表达下调及蛋白合成速率增加,这种作用与其降低DNMT1、DNMT3a所介导的ATRAP基因甲基化有关。4.Los早期干预长期抑制SHR LVH与ATRAP基因甲基化有关,提示表观遗传可能是EH“遗传性缺失”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 卤 0.3 vs 1.0 卤 0.0 , DNMT3a : 3.0 卤 0.3 vs 1.0 卤 0.0 , P0.05 ) . Compared with SHRUn group , the binding of DNMT3a to ATRAP gene significantly decreased ( DNMT3a : 0.9 卤 0.1 , 0.9 卤 0.1 , 1.0 卤 0.2 , 1.1 卤 0.2 , 1.1 卤 0.2 vs 3.0 卤 0.3 , P0.05 ) .
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1
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本文编号：2069838