【作者】 肖安；

【导师】 薛采芳； 裴建明； 郭海涛；

【作者基本信息】 第四军医大学 ， 病原生物学， 2007， 硕士

【摘要】 阿片肽和阿片受体在体内分布广泛,人们已经认识到其在中枢镇痛、心血管功能调节等方面都具有十分重要的作用。实验已经证明在心肌细胞膜和血管壁上存在大量的阿片受体,心脏亦可自身合成内源性阿片肽,提示阿片肽系统对心血管系统有自分泌调节作用。在体、离体的研究都发现阿片受体的激活具有抗缺血/再灌注性心律失常的作用,然而心肌缺血后心脏阿片受体系统如何变化,迄今尚无报道,对阿片肽和阿片受体的进一步研究将为临床应用阿片类物质治疗缺血性心律失常奠定理论基础。目的:(1)研究在缺血/再灌注不同时间点,大鼠心脏组织中κ阿片受体基因和蛋白质水平的表达。(2)给予κ阿片受体的选择性激动剂U50,488H和选择性阻断剂nor-BNI,研究κ阿片受体在抗缺血/再灌注大鼠心律失常中的作用。(3)采用Gi/o蛋白抑制剂、ATP敏感钾通道阻断剂、蛋白激酶C选择性抑制剂和酪氨酸激酶抑制剂,初步探讨κ阿片受体抗缺血/再灌注性心律失常可能涉及的细胞信号通路。方法:(1)实验采用健康雄性SPRAGUE-DAWLEY大鼠(体质量220～300 g),按随机原则分组,根据不同实验要求给予相应的处理。建立在体的大鼠冠状动脉缺血/再灌注动物模型,记录各项心功能指标,并在实验结束时分部位留取心肌组织。(2)运用RT-PCR技术检测在缺血/再灌注的不同时间点,大鼠心肌组织中κ阿片受体mRNA的表达水平。(3)运用WESTERN BLOT技术检测在缺血/再灌注的不同时间点,大鼠心肌组织中κ阿片受体蛋白质的表达水平。结果:1.缺血/再灌注不同时间点κ阿片受体基因水平和蛋白水平表达的变化情况在再灌注即刻、再灌注60 min、再灌注180 min时κ阿片受体mRNA表达量明显高于正常组和假手术组(P<0.01),而再灌注360 min时κ阿片受体mRNA表达恢复到正常水平;在再灌注即刻、再灌注60 min、再灌注180 min和再灌注360 minκ阿片受体蛋白质的表达量明显高于正常组和假手术组(P<0.05)。2.к阿片受体介导的抗心律失常作用对照组在观察期间内仅偶尔出现室性早搏,未发生室性心动过速和室颤。I/R组大鼠在心肌缺血时,心电图ST段明显抬高,可出现室性早搏、室性心动过速和室颤等心律失常;在再灌注即刻,又出现较为严重的室性心动过速和室颤,此后随着再灌注时间的延长,大鼠心电图ST段明显恢复,心肌缺血改善,心律失常的发生减少。U50,488H+I/R组大鼠在心肌缺血/再灌注期间,也可出现室性早搏、室性心动过速和室颤等心律失常,不过与I/R组相比,其持续时间明显缩短,发生率亦明显降低,心律失常的评分明显减少(P<0.01)。为进一步证实U50,488H的抗心律失常作用是否由к阿片受体所介导,在U50,488H+I/R组基础上,提前15 min给予大鼠选择性к阿片受体阻断剂nor-BNI,结果发现U50,488H的抗心律失常作用可被nor-BNI完全阻断(P<0.01);而nor-BNI本身对心肌缺血/再灌注所致心律失常没有影响(P>0.05)。3. Gi/o、PKC、KATP和TK阻断剂对к阿片受体抗心律失常作用的影响与I/R组相比,U50,488H+I/R组大鼠心律失常评分明显下降,若分别不同时间提前给予Gi/o蛋白抑制剂Pertussis toxin、KATP通道阻断剂glibenclamide和PKC的选择性抑制剂chelerythrine后,U50,488H的抗心律失常作用可以被显著减弱或完全阻断(P<0.05);而提前给予TK的抑制剂genistein对U50,488H的抗心律失常作用无明显影响(P>0.05)。结论:1.本研究首次发现心肌缺血/再灌注可导致心脏κ阿片受体的基因和蛋白质水平的明显上调,该反应有利于内源性阿片肽抗缺血/再灌注性心律失常作用的发挥,可能是心脏自我保护作用的一种代偿机制。2.心肌缺血/再灌注时,外源性к阿片受体激动剂U50,488H通过激动к阿片受体介导了明确的抗缺血/再灌注性心律失常的作用,该作用的细胞信号途径可能涉及Gi/o、PKC和KATP等分子。更多还原

【Abstract】 Endogenous opioid peptides (EOPs) and opioid receptors, which widely exist in diverse systems, play important roles in central analgesia as well as in the regulation of cardiovascular functions. Opioid receptors are found rich on the membrane of cardiac myocytes and in blood vessel wall,at the mean time, heart itself is found capable of producing EOPs, which suggests that opioid peptides act on the cardiovascular system. Our study showed that activation ofк-opioid receptor during myocardial ischemia and reperfusion in rats induced an anti-arrhythmic effect in vivo, as well as in vitro. However, it is not clear so far whether there is alters in the opioid receptor system under the condition of myocardial ischemia. Further study of EOPs and opioid receptors would hopefully build the theoretical foundation of treating arrhythmia induced by coronary artery ischemia with opioid in clinic.1. Objectives:(1) To observe the expression ofк-opioid receptor in myocardial ischemia and reperfusion in rats.(2) We use U50,488H (a selectiveк-opioid receptor agonist) and nor-BNI (a selectiveк-opioid receptor antagonist) to investigate the anti-arrhythmic effect ofк-opioid receptor during myocardial ischemia and reperfusion in rats.(3) To investigate the possible signal pathway involved in the anti-arrhythmic effect ofк-opioid receptor during myocardial ischemia and reperfusion by using pertussis toxin (a Gi/o protein inhibitor), glibenclamide (an ATP-sensitive potassium channel blocker), chelerythrine (a selective PKC inhibitor) and genistein (a Tyrosine kinase inhibitor).2. Methods:(1) Male Sprague-Dawley rats weighing 220～300 g were used for all experiments. The rats were randomly divided into different groups according to experiment protocol. Parameters of heart function were recorded during the myocardial ischemia and reperfusion injuries, and the tissue of heart was kept for afterwards analysis.(2) The RT-PCR technique was used to investigate the content ofк-opioid receptor mRNA in rat at different time point during ischemia and reperfusion.(3) The WESTERN BLOT technique was used to investigate the density ofк-opioid receptor protein in rat at different time point during ischemia and reperfusion.3. Results:(1) The changes ofк-opioid receptor gene and protein at the different time during ischemia and reperfusion:①compared with control group, the content ofк-opioid receptor mRNA was increased significantly at 0 min, 60 min and 180 min during reperfusion (P<0.01), and was decreased to the normal level at 360 min.②compared with control group, the density ofк-opioid receptor protein was increased significantly at 0 min, 60 min, 180 min and 360 min during reperfusion (P<0.05).(2) Anti-arrhythmic effects induced byк-opioid receptor activation: Few ventricular premature contractions were observed in the rats control group. After left anterior descending coronary artery (LAD) occlusion, ventricular premature contractions, ventricular tachycardia and ventricular fibrillation were examined by electrocardiogram. In the course of reperfusion, much more ventricular arrhythmia appeared especially at the immediate time of reperfusion. Finally, the incidence of ventricular arrhythmia was reduced with the development of reperfusion. With the pretreatment of U50,488H, the endurances of ventricular arrhythmia in the rats of U50,488H+ischemia/reperfusion (U50,488H+I/R) group were significantly shortened, and the incidence of ventricular arrhythmia was reduced as well as the arrhythmia score (P<0.01). With the pretreatment of nor-BNI for 15 min in U50,488H+I/R group, the anti-arrhythmic effect of U50,488H was completely blocked (P<0.01). But it had no effect on the arrhythmia induced by ischemia and reperfusion in I/R group (P>0.05).(3) The influence of inhibitors of Gi/o protein, PKC and Tyrosine kinase, ATP-sensitive potassium channel blocker on the anti arrhythmic effect ofк-opioid receptor activation: Pretreated with pertussis toxin, glibenclamide and chelerythrine respectively, the anti-arrhythmic effects induced by U50,488H during myocardial ischemia and reperfusion were significantly attenuated or even were completely blocked (P<0.05). The anti-arrhythmic effects of U50,488H were not significantly affected by pretreatment with genistein(P>0.05).4. Conclusions(1) Our study finds that the increasing expression ofк-opioid receptor is induced by the myocardial ischemia and reperfusion for the first time. And it is helpful for the EOPs to act the anti-arrhythmia effect during myocardial ischemia and reperfusion. This may be a compensational mechanism of heart’s self-protection.(2) U50,488H exerts an anti-arrhythmic effect through activatingк-opioid receptor in the rats with myocardial ischemia and reperfusion. The signal pathway may be correlated with Gi/o, PKC and KATP channel.更多还原