【作者】 宋康兴；

【导师】 王士雯； 卢才义；

【作者基本信息】 中国人民解放军军医进修学院 ， 老年心血管病， 2009， 博士

【摘要】 背景:脑利钠肽(BNP)在心血管疾病中的应用日趋广泛,有可能成为一项心血管疾病预后判断和死亡危险分层的“生化标志物”。有证据显示BNP血浆浓度与心房颤动(AF)有密切的联系,AF时BNP和肾素-血管紧张素-醛固酮系统(RAs)均参与神经内分泌调节,但两者的联系及其意义尚不明确。目的:观察快速心房起搏对实验犬血浆和心肌组织BNP的影响,分析BNP与RAS的变化,探讨两者在神经内分泌和心肌组织局部调控中的作用和相互联系,明确BNP对于心房颤动评估中的意义。方法:经右侧颈外静脉植入电极建立心房快速起搏(RAP)犬的动物模型,RAP组(n=5)给予400次/分高频电刺激6h,而假手术组(Sham)不给予电刺激(n=6)。分别于0h、1h、2h、4h、6h取血,ELISA法测血浆BNP、血管紧张素Ⅱ(AngⅡ)和醛固酮(ALD);快速心房起搏6h后取左心室游离壁、右心室游离壁、右心房侧壁和左心房侧壁组织标本,用免疫组织化学法(IHC)和免疫荧光法观察BNP在心肌组织中的分布与表达,免疫印迹法(WesternBlot)测定心肌组织BNP浓度,逆转录-PCR法(RT-PCR)检测BNP、A型钠尿肽受体(NPR-A)、B型钠尿肽受体(NPR-B)、血管紧张素原(AGT)、血管紧张素转化酶(ACE)的mRNA表达。结果:快速心房起搏犬血浆BNP水平迅速升高,并随起搏刺激的存在而维持在较高水平(1h:45.3±14.6 VS 23.0±6.9 pg/ml;2h:71.5±24.5 VS28.7±13.1;4h:76.5±15.3 VS 32.5±8.3:6h:85.7±29.1 VS 85.7±29.1。与Sham组比较,p均＜0.01);AngⅡ水平则在2h之后出现升高(2h:88.6±22.2 VS 47.7±11.2 pg/ml;4h:102.18±35.8 VS 46.1±15.7;6h:104.94±13.6 VS 47.1±21.2,与Sham组比较,P均＜0.01)。血浆BNP水平与AngⅡ水平呈明显正相关(r=0.737,P＜0.01)。血浆ALD水平的变化无统计学差异。免疫组化染色和免疫荧光染色结果显BNP表达主要位于胞浆内,可见局灶性聚集现象。激光共聚焦显微镜观察到RAP组心房有较强的BNP表达,以右心房最为显著;RAP组左心室和右心室组织BNP染色强度均显著高于Sham组(0.61±0.06 VS 0.29±0.13;0.47±0.09 VS 0.32±0.11,P均＜0.05)。Western Blot显示RAP组心房、心室的BNP蛋白水平均显著高于对照组(P均＜0.05)。RAP组心房的BNP水平高于其心室,且右心室BNP蛋白水平显著高于左心室。与Sham组相比:RAP组左心室BNP mRNA下调(P＜0.05),而右心室BNP mRNA上调(P＜0.01);左右心室NPR-A mRNA均下调(P均＜0.01);心室和心房肌NPR-B mRNA上调(P均＜0.05);AGT mRNA在右心房则上调(P＜0.01),在其它心房心室则下调(P＜0.05,P＜0.01);左心室、左心房的ACE mRNA表达下调(P＜0.05,P＜0.01),而右心房和右心室的ACE mRNA上调(P＜0.01,P＜0.05)。结论:快速心房起搏数小时即可引起血浆和心肌组织BNP的变化,BNP对AF的评估有重要意义。快速心房起搏后BNP在心房和心室的表达均增加,且心房的BNP水平高于心室,心房是BNP的主要来源。右心房BNP mRNA增加并与AGT、ACE共表达,提示BNP和RAS除通过内分泌途径发挥调节作用外,也作为局部自分泌和/或旁分泌因子参与急性心房颤动的调节。更多还原

【Abstract】 Background Previous studies have suggested extensive applications of brain natriuretic peptide(BNP) in cardiovascular diseases.There is increasing evidence showing that BNP is related to atrial fibrillation(AF) and it may be a potential predictor of AF.Both BNP and renin-angiotensin-aldosterone system (RAS) play important roles in AF regulation,but their correlations have not been studied clearly,particularly in blood and cardiac tissue.Objective To study the effect of rapid atrial pacing(RAP) on BNP and RAS, as well as their correlations,furthermore,to analyse the role of BNP in acute AF assessment.Methods Acute AF model was established by implanting electrodes through canine right external jugular vein,RAP group(n=5) received continuous pacing of 400 beats per min(bpm) for 6 hours,while sham group(n=6) underwent no electronic stimulation.Serum BNP,angiotensinⅡ(AngⅡ) and aldosterone(ALD) were tested by enzyme linked immunosorbent assay(ELISA) on 0h,1h,2h,4h and 6h after pacing.At the time point of 6h,samples from free wall of right ventricle(RV) and left ventricle(LV),lateral wall of right atrium(RA) and left atrium(LA) were collected,distribution and expression of BNP in them were observed by immunohistochemical method(IHC) and immunofluorescence. Protein level of BNP was tested by Western Blot.Furthermore,mRNA of BNP, natriuretic peptide receptor-A(NPR-A),natriuretic peptide receptor-b(NPR-B), angiotensinogen(AGT) and angiotensin converting enzyme(ACE) were measured by reverse transcription PCR(RT-PCR).Results The BNP level was significantly increased after RAP in the pacing group(1h:45.3±14.6 pg/ml VS 23.0±6.9 pg/ml;2h:71.5±24.5 pg/ml VS 28.7±13.1 pg/ml;4h:76.5±15.3 pg/ml VS 32.5±8.3 pg/ml;6h:85.7±29.1 pg/ml VS 85.7±29.1 pg/ml,respectively,all P＜0.05 vs.Sham):AngⅡincreased since 2h after pacing(2h:88.6±22.2 pg/ml VS 47.7±11.2 pg/ml:4h:102.18±35.8 pg/ml VS 46.1±15.7 pg/ml;6h:104.94±13.6 pg/ml VS 47.1±21.2 pg/ml,respectively, P＜0.01 vs.Sham).Regression analysis revealed a positive correlation between Serum BNP and AngⅡ(r=0.737,P＜0.01 vs.Sham).Serum ALD showed no obvious changes in 6h after RAP.IHC and immunofluorescence observation showed BNP positive grains and some focal aggregation in intracytoplasm.After RAP,the intensity of positive dyeing turned to be more intensive in atrium and ventricle,especially in right atrium.Significant difference was noted in the desity of positive BNP dots(0.61±0.06 VS 0.29±0.13;0.47±0.09 VS 0.32±0.11,both P＜0.05 vs Sham).Western Blot showed that RAP group had higher atrial and ventricular BNP levels than sham group(P＜0.05).RAP group had decreased LV BNP mRNA(P＜0.05 vs.Sham) and increased BNP mRNA in RV and RA; NPR-A mRNA downregulated in ventricles(P＜0.01 vs.Sham) while significant NPR-B mRNA upregulation was observed in cardiocyte(LV P＜0.01 vs.Sham, atria and RV P＜0.05 vs.Sham);significant AGT mRNA expression was observed only in RA(P＜0.01 vs.Sham);ACE mRNA downregulated in LA and LV(P＜0.05 vs.Sham,P＜0.01 vs.Sham),while it increased both in RA and RV(P＜0.01 vs.Sham,P＜0.05 vs.Sham).Conclusion Short-term RAP results in rapid changes of BNP in blood and myocardium tissue,and there is parallel expression of AGT mRNA,ACE mRNA and BNP mRNA in right atrium.There may be a relationship between BNP and AngⅡindicating BNP and RAS act as autocrine or paracrine secretion factors in local tissue regulation except for its neruocrine functions.BNP may be a useful predictor of acute AF.更多还原