【作者】 肖斌；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 目的与意义高血压是严重危害我国人民健康的常见病和多发病,具有发病率高,致残率高,死亡率高的特点。高血压是一种遗传、环境等多因素参与的全身性疾病,其发病机制很复杂,但遗传因素起着十分重要的作用。药物治疗有一定的疗效,但需要终身服药,且由于多方面的原因患者常常难以坚持正规治疗;因此,寻求更为有效、经济、简单的治疗方法成为一项研究的重要课题。重组腺相关病毒载体(rAAV)系统由于能携带基因转染分裂期和非分裂期细胞(即具有广泛的转基因范围)、无副作用(无免疫源性)、感染效率高、能驱动基因在体内长期表达,为高血压病的基因治疗提供了新的手段。由花生四烯酸(arachidonic acid, AA)通过细胞色素P450(cytochrome P450,CYP)表氧化酶途径代谢产生。AA是人体内多种重要心血管活性物质的前体,主要是以酯化的形式存在于细胞膜的内表面,在脂解激素作用下可由磷脂酶A2水解释放至细胞浆内。对AA的研究已有多年,最为人们熟知的是环氧化酶和脂质氧化酶代谢途径,近年研究发现还可通过细胞色素P450途径代谢(即AA代谢的第三条途径),包括AA-CYP表氧化酶和ω-羟化酶,其中AA经表氧化酶代谢产生四种EETs (5,6-, 8,9-, 11,12-和14,15-EET),研究发现EETs可以通过激活钙离子敏感的钾通道,使平滑肌细胞处于超极化状态而扩张血管。CYP表氧化酶包括2C和2J两类,研究显示CYP表氧化酶在人类心脏和血管内皮细胞中有丰富表达。花生四烯酸CYP450表氧化酶及其代谢产物EETs在心血管系统的作用逐渐为人们所重视,在离体和体外实验中, EETs被证实在血管的扩张和高血压的调控方面有着重要的作用,然而在在体水平的研究还没有充分的依据。由此我们将在在体水平研究表氧化酶对SHR大鼠的血压调节作用。研究其能否阻断高血压的进展,其机制又是什么。表氧化酶基因过表达与其他血管活性物质之间又有何内在联系?本实验将从以下几个方面进行探讨:1通过重组腺相关病毒载体将CYP450表氧化酶2J2基因导入到自发性高血压大鼠体内,观察表氧化酶基因2J2在动物体内长期稳定的表达情况;2观察CYP450表氧化酶2J2基因的过表达对SHR血压的影响及机制;3观察CYP450表氧化酶2J2基因的过表达对SHR心功能和血管功能的影响及机制;4观察CYP450表氧化酶2J2基因的过表达对其他血管活性物质的影响。实验方法1.动物分组:雄性自发性高血压大鼠SHR20只,体重在220±10 g,三次尾动脉压测定均>160mmHg,随机分为5组,即Control组,GFP组,F87V组,2J2组,每组5只,分别予以病毒溶解液,rAAV-GFP, rAAV-F87V, rAAV-2J2,病毒尾静脉注射,病毒量为1×1012pfu/只;2.采用三质粒共转染法包装重组腺相关病毒;3. Elisa试剂盒检测尿中14,15-DHET的含量;4.应用western blot检测蛋白的表达;5.血液动力学检测:动物用2%戊巴比妥钠腹腔注射麻醉(1mL/kg),将Millar管经右侧颈动脉插管送入左心室,检测左心室的功能,数据由MPVS-300(Millar Pressure-Volume System,美国Millar Instrument PowerLab公司)系统采集并分析。5.血管张力测定采用多导生理记录仪记录;7.实时PCR检测心肌ANP及ANP-C型受体(NPR-C)mRNA的表达;8. ANP在心肌细胞的表达及分布情况采用免疫组化DAB染色法;9.原代心肌细胞培养采用胶原酶消化法。实验结果:1 CYP450表氧化酶2J2基因在SHR体内的表达情况14,15-DHET是14,15-EET的代谢产物,能反应EETs在体内的代谢水平,我们通过14-15-DHET的Elisa试剂盒检测尿中的14-15-DHET水平,发现在转表氧化酶基因2J2大鼠的14-15-DHET水平较对照组提高越11倍。应用Western blot的方法检测心肌组织中2J2表达水平,证实2J2在心肌中有表达。2 CYP450表氧化酶2J2基因对动脉压的影响在实验过程中对鼠尾动脉血压进行动态监测,发现从第2月开始,转染表氧化酶基因组血压在第2月时血压从初始血压173.0±2.8mmHg降至163.5±5.8mmHg,而对照组从初始血压174±3.9mmHg升至179.0±4.9mmHg,至6月时转2J2组血压维持在165.0±4.7 mmHg,而对照组继续升至195.2±20.8mmHg。在处死动物前,从颈动脉进行有创测压,发现转2J2组血压较对照组显著下降(191.0±25.1 mmHg vs. 226.8±25.8mmHg, p<0.01)。3 CYP450表氧化酶2J2基因心功能的影响我们对各组大鼠进行了心功能的测定,其结果显示,转染表氧化酶基因大鼠的心内最大收缩压(201.8±32.8 mmHg)与对照组(245.9±16.2 mmHg)相比较显著下降,收缩末压(End-systolic Pressure)从对照组的237.6±14.2 mmHg降至199.0±33.3mmHg,dp/dt max从17005.5±1850.8mmHg/s降至13223.4±1792.4 mmHg/s,dp/dt min的绝对值从对照组的16930.8±2080.0mmHg/s降至-12258.1±4155.6mmHg/s。这些指标显示心脏的收缩功能明显下降,但是,有趣的是转染表氧化酶基因大鼠的心搏出量(Stroke Volume)却没有因心肌收缩功能降低而下降,与对照组96.3±14.9 uL相比较,增加至137.9±20.9uL ;心输出量(CO)从对照组的19826.1±2906.0μL/min增加到26870.7±6431.2μL/min,而心率无显著变化。4 CYP450表氧化酶2J2基因对血管功能的影响血管弹性回缩率(Effective arterial elastance,Ea )是反应血管弹性的指标之一,转染表氧化酶基因组的Ea值较对照组显著降低,从2.6±0.2降低到1.5±0.37。我们还取大鼠胸主动脉环行血管收缩舒张实验,发现转染了表氧化酶2J2基因大鼠的动脉环对肾上腺素的收缩反应较对照组显著降低,并显著增强了血管对Ach的舒张反应。5对心房利钠肽ANP及其C-型受体的影响我们取转表氧化酶基因的组织,对心房利钠肽ANP及利钠肽C-型受体NPR-C、内皮素ET1及内皮素1-型受体EdnrA、肾上腺皮质素ADM及其受体ADMR的mRNA水平通过real-time PCR进行了定量检测,我们惊喜的发现,心脏ANP的mRNA表达水平与对照组相比较约有14倍的上调,其C型受体(NPR-C)在心、主动脉、肾脏和肺脏中亦明显上调,而ET1及受体EdnrA、ADM及其受体ADMR mRNA的水平无显著变化。在明确了表氧化酶在心肌mRNA表达上调后,我们对心肌组织中的ANP蛋白质水平用Western blot的方法进行了检测,发现心肌中ANP蛋白质表达亦随之上调。为了进一步明确ANP的表达在心肌组织中的分布情况,我们应用ANP的一抗作了心肌组织的免疫组化染色,发现转染表氧化酶基因大鼠心脏阳性细胞明显增多,并主要分布于心内膜下心肌组织。6外源性EETs对培养心肌细胞ANP的影响我们取乳鼠的心肌细胞作体外培养,予以外源性EETs进行干预,发现11,12-EET和14,15-EET能够显著刺激心肌细胞ANP mRNA和蛋白水平的上调,而且呈现剂量依赖性。结论本实验研究通过体内和体外实验,利用血压动力学和分子生物学技术,对表氧化酶及其代谢产物EETs在高血压方面的调节机制作了一系列的研究,得出了如下结论:1重组腺相关病毒能有效介导外源性基因在体内的长期稳定的表达,是实验研究中一个良好的基因导入载体,也是一种临床疾病基因治疗的理想手段;2表氧化酶及其代谢产物EETs能有效降低血压,遏制高血压病的进展,其机制如下:(1)表氧化酶及其代谢产物EETs对心肌的负性肌力调节作用可能是其降低高血压的重要机制之一,但这种对心肌的负性肌力调节并不以减少心脏的每搏量和心输出量为代价;(2)表氧化酶及其代谢产物EETs上调心脏ANP的合成,再通过ANP的生物活性来扩张血管,抑制心脏收缩力和利尿作用来调节血压,这可能是表氧化酶及其代谢产物EETs对血压进行调节的补充机制之一;(3)表氧化酶及其代谢产物EETs能有效降低血管对儿茶酚胺的反应性,并增强对舒血管物质的反应,从而有效舒张血管,参与了血压的调节;(4)表氧化酶基因的过表达能够显著增加中心大动脉的弹性和顺应性,增加了大血管对心脏射血的缓冲能力,有效降低收缩压。其机制可能与表氧化酶及其代谢产物EETs促进心脏ANP的分泌及上调主动脉和心脏的NPR-C,从而抑制纤维细胞增生,减轻血管硬化和心肌重构有关。更多还原

【Abstract】 Background:Epoxyeicosatrienoic acids (EETs), which are cytochrome P450 (CYP) epoxygenase derived metabolites of arachidonic acid(AA), display many of the characteristics of endothelium-derived hyperpolarizing factor (EDHF) and cause hyper polarization of underlying vascular smooth muscle cells via activation of Ca2+ activated K+ channels. So EETs are thought to be potential candidates for EDHF. It was demonstrated that Arachidonic acid cytochrome P450 epoxygenase metabolites, EETs, have potent vasodilatory and diuretic feature, and therefore have hypotensive effect. No in vivo studies, however, were performed to support it. In the current study, we investigated this hypothesis via overexpressing CYP epoxygense genes in spontaneously hypertensive rats (SHR).Methods and Results:1) The recombinant adeno-associated virus vector (rAAV) was utilized to mediate long-term transfection of CYP2J2 genes in adult SHR, and animal systolic blood pressure was monitored using arterial caudilis indirect manometric method and main stable hydrolysis metabolic product of 14, 15-EET, 14, 15-DHET, in urine was measured to reflect the production of EETs. Results showed that at 2 months the urinary excretion of 14, 15-DHET increased by 11-folds in the rAAV-2J2 groups compared with rAAV-GFP-treated group. 2) The systolic blood pressure in rAAV-2J2-treated group decreased from 173.0±2.8mHg to 163.5±5.8mmHg ( p﹤0.01) at month 2, and it is 165.0±4.7 mmHg at month 6 after gene injection. In contract, in rAAV-GFP treated control animals, the systolic blood pressure was kept higher (from 174±3.9mmHg to 176.7±5.3mmHg) at month 2 and increased to 195.2±20.8mmHg at month 6 (~30mmHg higher than that in rAAV-2J2-treated group, p<0.001). 3) Before the rats were sacrificed at month 6, the blood pressure form carotid artery was detected with Pressure-Volume System. The artery blood pressure of 2J2-treated group was decreased to 191.0±25.1 mmHg (vs. 226.8±25.8mmHg, p<0.01). At the same time, the cardiac functions were measured. The results showed that maximum intra-cardiac pressure was 202.1±30.0 mmHg in 2J2-treated group, significantly lower than control (vs. 241.2±18.2 mmHg , p﹤0.01). The end-systolic pressure in treated group was 199.0±33.3mmHg, and that in the control group was 237.6±14.2 mmHg. Other valuable indexes for evaluating the myocardial contractility, dp/dt max and dp/dt min, were also measured. Both the dp/dt max and the absolute value of dp/dt min were decreased from 17005.5±1850.8mmHg/s and 16930.8±2080.0mmHg/s in control group to 16930.8±2080.0mmHg/s and 12258.1±4155.6mmHg/s in 2J2-treated group, respectively. However, the cardiac output (CO) in treatment animals was significantly higher than control (26870.76431.3μL/min vs. 19826.1±2906.0μL/min, p<0.05). 4) Recorded arterial elastance (Ea) index in the rAAV-2J2 agroup was 1.5±0.4 (vs.2.6±0.4, p﹤0.001), which demonstrated that the overexpression of the CYP2J2 gene attenuates the arterial sclerosis. The responsiveness to norepinephrine (NE) and acetylcholine (Ach) of the thoracic aorta rings was checked and it was demonstrated 2J2 gene could attenuate the reactivity of the rings to NE, and enhance the diastole respond to Ach. 5) Interestingly, we found the atrial natriuretic peptide (ANP) mRNA were up-regulated 14 folds in myocardium of the rAAV-2J2 group; furthermore, the C-type receptor’s (NPR-C) mRNA of ANP was increased in heart, aorta, kidney and lung tissues. However, the expression levels of some other vasoactive substances such as endothelin-1 and its receptors, adrenomedullin and its receptors have no change detectable. 6) Finally, we cultured the primary myocardium cell to study effect of EETs on ANP level, and we also found addition of exogenous EETs up-regulated the protein of ANP and had a dose-effect relationship.Conclusion:For first time our study indicates transfection of rAAV-mediated 2J2 genes could decrease the blood pressure and prevent development of hypertension in SHR. Its mechanisms are including: 1) CYP epoxygenase and its metabolites EETs mediate negative inotropic effect on the cardiac contractility., which is one of their mechanism on blood-pressure regulation. On the same time, the reduction in cardiac contractility didn’t cause to decrease in the cardiac output. In contrast, cardiac output was increased. The negative inotropic effect with increasing CO would be valuable for the 2J2-transgene application to clinic. 2) Except for the direct effects of their metabolites EETs on the myocardium and vessels, CYP2J2 may provide beneficial effects via up-regulating expression of ANP, which can depress the cardiac contractility and produce vasodilatation and natriuresis. The signal mechanism may also play an important role in regulation of blood pressure. 3) CYP2J2 gene could attenuate the reactivity of the rings to norepinephrine, and enhance the diastole respond to acetylcholine. The effect would benefit the regulation of CYP2J2 gene on blood pressure. 4) The overexpression of CYP2J2 gene can decline the Ea of the aorta and induce a larger increase in aorta compliance. The function of CYP2J2 gene’s attenuating the arterial sclerosis and improving its relaxation should partly attribute to ANP and NPR-C, which were up-regulated by CYP2J2 gene.更多还原