【作者】 张鹏；

【导师】 朱运龙；

【作者基本信息】 第四军医大学 ， 生理学， 2004， 博士

【摘要】 目的：研究在心肌缺血再灌注损伤过程中，κ阿片受体介导的心脏保护作用以及抗心律失常作用机制；初步探讨激活κ阿片受体对血浆肌酸磷酸激酶（CK）、乳酸脱氢酶（LDH）以及血管紧张素Ⅱ（AngⅡ）、内皮素（ET）和一氧化氮（NO）等体液因子的调控作用。 方法：实验采用健康雄性SD大鼠（体重250～350g），按随机原则分组，每组8只大鼠，根据不同实验要求给予相应的处理。心肌缺血再灌注损伤大鼠动物模型：用30g·L^-1戊巴比妥钠麻醉(1.5ml·kg^-1, ip大鼠，气管插管，微型动物呼吸机给予正压人工呼吸，分离左侧颈静脉建立给药途径。而后于大鼠胸骨左缘第4～5肋间开胸，暴露心脏，剪开心包膜，在冠状动脉左前降支（LAD）距左心耳下缘约2 mm处穿线（5／0丝线），再用直径约0.2 cm细塑料管穿过丝线，以备结扎，稳定15 min后再进行下一步实验。心肌缺血时用止血钳夹紧结扎线，再灌注时只需将结扎线松开即第四军医大学博士学位论文可。以心电图ST段明显抬高、结扎线以下心肌颜色变苍白、左心室内压迅速下降作为急性心肌缺血标志。实验过程中记录大鼠肢体H导联心电图、股动脉压（ABP）以及左心室内压（LVP）及心脏收缩（dP/d tmax）和舒张功能（一dP/dtmax）等血流动力学指标。实验结束后采集大鼠心肌组织和血清样本，观察不同组别大鼠心肌超微结构、心肌梗死面积等指标的变化，并检测大鼠血浆肌酸磷酸激酶（CK）、乳酸脱氢酶（LDH）以及血管紧张素n（Angn）、内皮素（ET）、一氧化氮（NO）等体液因子水平。结果:1.血流动力学指标 （l）选择性K阿片受体激动剂U50488H在较小剂量时（0.5mgkg一’）对大鼠血流动力学指标影响较小，与对照组相比无显著差异(尸>0.口的;而u50488H在较大剂量时（l .5 mgk扩），可明显降低大鼠心率（HR）、动脉压（ABP）、左心室内压（LVP）及收缩（+dP/d tmax）和舒张功能（一dP/d tmax），与对照组相比差异显著（尸<口.口了）。 （2）与对照组相比，大鼠在给予 U50488H后，短时间内即出现心率减慢，动脉压、左心室内压及收缩和舒张功能下降（尸<口.0了），上述指标在随后的观察时间内变化不大。1瓜组大鼠在给予心脏缺血处理后，心率无明显变化;动脉压、左心室内压以及心脏收缩和舒张功能则明显下降（尸<ao]），再灌注时大鼠动脉压、左心室内压以及心脏收缩和舒张功能有所回升，但仍未能达到心脏缺血前水平。U50488H+I瓜组大鼠在给予U50488H后，心率变慢，动脉压、左心室内压、心脏收缩和舒张功能下降 （尸<ao!），进一步给予心脏缺血处理后，上述指标虽继续下降，但与缺血前比较无显著性差异(P>0.口助，再灌注时亦无明显回升。提前给予选择性K阿片受体阻断剂NO卜BNI能够抑制U50488H的上述作用。第四军医大学博士学位论文 2.心肌超微结构变化 对照组和U50488H组大鼠心肌纤维排列整齐，肌节各带清晰，线粒体包膜完整，峭密集，内质网无扩张，微小血管管腔开放。皿组和BNI+U50488H+I/R组大鼠心肌纤维肌丝闰盘增宽，其它各带模糊，部分线粒体膜破损，线粒体肿胀、溶解，晴紊乱，内质网呈空泡化，小血管管腔内有微血栓形成。U50488H+I瓜组大鼠心肌纤维结构基本正常，肌丝完整、清晰，线粒体稍肿胀，内质网空泡化少见，小血管管腔开放，偶可见微血栓。 3.心肌梗死面积 在给予心肌缺血45 min，再灌注120 min后，FR组大鼠缺血心肌出现明显的心肌梗死现象;与1/R组大鼠心肌梗死面积相比较，U50488H+FR组大鼠心肌梗死面积明显缩小（P<0.0!）;BN卜U50488H十I瓜组大鼠与I瓜组大鼠心肌梗死面积无显著性差异（尸>口.口J），但与U50488H+I瓜组大鼠心肌梗死面积相比差异显著（P<0.01）。 4.K阿片受体介导的抗心律失常作用及机制 （1）K阿片受体介导的抗心律失常作用 对照组和U50488H组大鼠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很组大鼠在心肌缺血时，心电图ST段明显抬高，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，又可出现较为持久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。U50488H+FR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1/R组相比，其持续时间明显缩短，发生率亦明显降低（尸<0.01）。 （2）K阿片受体介导的抗心律失常作用机制 ①提前给予大鼠腹腔注射利血平（lm叭g，1次/天，共3第四军医大学博士学位论文天），结果发现U50488H的抗心律失常作用明显减弱（尸<0.01）;提前给予大鼠静脉注射阿托品（lm叭g），U50488H的抗心律失常作用无明显变化（P>口.仍）。 ②与U50488H+I/R相比，提前给予Gi/o蛋白抑制剂pertussistoxin（48h预处理，10 pg瓜g）、No合成酶抑制剂L一NAME（50mg/kg，在给予U50488H前15 min）、K二P通道阻断剂glibenelam记e （0.3 mg/kg，在给予U50488H前30 min）和PKC的选择性抑制剂ehelerythrine（5 mg/kg，在给予uso4ssH前5 min），结果发现U50488H的抗心律失常作用减弱（尸<0.01）;而提前给予TK的抑制剂gen更多还原

【Abstract】 Objective: To investigate Cardioprotection mediated by -opioid receptor and underlying anti-arrhythmic mechanism in rats subjected to myocardial ischemia/reperfusion injury and to discuss the modulating effects of K -opioid receptor activation on the contents of CK, LDH, AngII, ET and NO in plasma.Methods: Male Sprague-Dawley rats weighing 250~350g were used for all experiments. The rats were randomly divided into different groups according to experiment protocol, they were control group, ischemia/reperfusion (I/R) group, U50488H group, U50488H+ I/R group and Nor-BNI+U50488H+ I/R group. Animal model of myocardial ischemia and reperfusion injuries: The surgical procedures used here were similar to those previously employed by others. The rats were anesthetized with ip injection of 30 mg-kg-1bentobarbitone and supplemental doses were given i.v. when necessary to ensure a adequate level of anesthesia. The trachea was cannulated and all rats were artificially ventilated. The right jugular vein was cannulated for injection of drugs. After thoracic cavity was opened, a 5/0 silk thread was passed around the left anterior descending coronary artery (LAD) with a taper needle, and the ends were passed through a small vinyl tube to form a snare. The acute myocardial ischemia induced by LAD occlusion resulting from pulling the snare was confirmed by regional cyanosis and a substantial fall in left ventricular pressure. Reperfusion was achieved by releasing the snare. Electrocardiogram, heart rate (HR), arterial blood pressure (ABP), left ventricular pressure (LVP), contractive function (+dp/dtmax) and diastolic function (-dp/dtmax) were monitored in the course of experiment. When the experiment was over, bloods of rats were gathered to measure the contents of CK, LDH, Ang II , ET and NO in plasma. Finally, the rats were killed and the hearts were used for the examination of the myocardial ultra-structures and the measurement of the infarction sizes.Results: 1. Hemodynamics data:(1) In comparison with control group, U50488H elicited no effect on hemodynamics in rats at low dose (0.5 mg-kg-1) (P>0.05), While at dose of 1.5 mg-kg-1, HR, ABP, LVP and 卤dp/dtmax of rats were significantly decreased with the administration of U50488H(2) Compared with control group, no change occurred in HR of rats in I/R group during I/R (P>0.05). While ABP, LVP and 卤 dp/dtmax in the rats with ischemia/reperfusion injury were loweredafter occlusion of LAD (P<0.01). During reperfusion, these indexes were restored, but were still lower than those before ischemia. In U50488H+ I/R group, with the administration of U50488H only, the cardiac functions were immediately decreased (P<0.01). After the further disposal of I/R, no further decrease was observed (P>0.05). The negative inotropic effects of U50488H were abolished in the presence of Nor-BNI.2. Myocardial ultra-structures:Similar to control group, there was no significant change of the myocardial ultra-structures in U50488H group. The structures of cardiac muscle fiber and endoplasmic reticulum were in order, the lumens of capillary were open. But myocardial ultra-structures in I/R group and BNI+U50488H+ I/R group were messed up, and there were a few of microthrombus in the lumens of capillary. Following U50488H intravenously injected 15 min before I/R, the injuries of the myocardial ultra-structure caused by ischemia/reperfusion were attenuated significantly.3. Myocardial infarction size:In the rat hearts of I/R group, there appeared obvious myocardial infarction. With the administration of U50488H in advance, the myocardial infarction size was reduced (P<0.01). The effect of U50488H was, although not completely, abolished by Nor-BNI (P<0.01).4. Anti-arrhythmic effects and mechanisms mediated by -opioid receptor:(1) Anti-arrhythmic effects mediated by -opioid receptor in rats subjected to myocardial ischemia/reperfusion injury:Similar to control group, few ventricular premature contractionswere observed in the rats of U50488H group, but no ventricular tachycard更多还原