【作者】 张冬梅；

【导师】 常业恬；

【作者基本信息】 中南大学 ， 麻醉学， 2009， 博士

【摘要】 目的①研究舒芬太尼预处理对缺血再灌注大鼠心肌是否具有保护作用;②研究舒芬太尼预处理对缺血再灌注大鼠心肌细胞凋亡的影响,探讨Bcl-2和Bax基因在舒芬太尼预处理中的调控作用;③研究纳洛酮、蛋白激酶C阻断剂对舒芬太尼预处理心肌缝隙连接蛋白Cx43及其mRNA表达的影响,初步探讨阿片受体、蛋白激酶C及Cx43是否介导了舒芬太尼预处理的心肌保护作用。方法实验分三部分:①健康雄性SD大鼠,体重250～350g,采用结扎左冠状动脉前降支法制备心肌缺血再灌注模型。随机分为6组(n=8),假手术组(S组)只穿线,不结扎左冠状动脉前降支;缺血再灌注组(I/R组)缺血30 min,再灌注120 min;缺血预处理组(IPC组)缺血5 min,再灌注5 min,重复3次后同I/R组;LS组、MS组和HS组分别给予舒芬太尼0.25、1.0、5.0μg/kg,持续5 min,停5 min,重复3次后同I/R组(总量为0.75、3.0、15.0μg/kg)。记录缺血再灌注前后不同时间点心功能指标,检测心律失常发生率并做出评分;TTC染色测定心肌梗塞面积,检测血清丙二醛(MDA)浓度及超氧化物歧化酶(SOD)活性;②根据第一部分结果,分别采用TUNEL法和免疫组织化学法检测S组、I/R组、IPC组及SPC组(舒芬太尼预处理3.0μg/kg)心肌细胞凋亡指数,心肌组织中凋亡相关蛋白Bcl-2、Bax的表达水平,以平均光密度(OD)反映其表达量;③在第二部分基础上,分别给予阿片受体阻滞剂纳络酮(Nal组)或联合舒芬太尼(Nal+SPC组),蛋白激酶C阻断剂白屈菜红碱(Che组)或联合舒芬太尼(Che+SPC组),免疫组织化学法检测心肌Cx43的分布,半定量分析每个视野下阳性反应的平均光密度(OD),RT-PCR技术检测心肌组织中Cx43 mRNA相对表达量。结果①与S组比较HR、MAP、LVDP及±dp/d tmax组间比较差异无统计学意义(P＞0.05),但I/R组各指标多低于S组;与缺血前即刻(T0)比较,I/R组在缺血再灌注后不同时间点MAP、LVDP及+dp/d tmax有不同程度的明显降低(P＜0.05或P＜0.01);+dp/d tmax在LS组再灌30min、120min低于T0(P＜0.05)。IPC和舒芬太尼预处理各组LVDP及±dp/d tmax在再灌注30min后多逐渐回升,与T0比较无统计学意义(P＞0.05)。IPC组及舒芬太尼预处理各组心律失常评分、梗死范围(IS/MR)与I/R组比较显著降低(P＜0.01)。舒芬太尼预处理各组MDA明显低于I/R组(P＜0.05),H组MDA较S组低(P＜0.05),SOD则高于I/R组,但明显低于S组(P＜0.05);②S组细胞凋亡指数低,I/R组细胞凋亡指数明显上升(P＜0.01),IPC组和SPC组仍可见凋亡细胞,细胞凋亡指数介于S组与I/R组之间,高于S组而低于I/R组,组间比较差异有统计学意义(P＜0.01)。与S组比较其余三组Bcl-2、Bax蛋白表达OD值明显升高(P＜0.01)。IPC组和SPC组Bcl-2蛋白表达OD值明显高于I/R组,Bax蛋白表达则低于I/R组,两组的Bcl-2/Bax比值明显高于I/R组(P＜0.01);③与I/R组比较,缺血预处理和舒芬太尼预处理上调Cx43蛋白表达量及mRNA水平(P＜0.05 or P＜0.01),在Nal+SPC组和Che+SPC组,纳洛酮和白屈菜红碱阻断了舒芬太尼预处理的作用,使Cx43蛋白表达量及mRNA水平未发生明显改变(P＞0.05)。结论1.舒芬太尼预处理对缺血再灌注大鼠心肌具有保护作用,其降低大鼠心律失常的严重程度,限制梗塞范围的作用,以3.0、15.0μg/kg剂量组的作用效果更佳;2.舒芬太尼通过上调Bcl-2蛋白表达,下调Bax蛋白表达,提高Bcl-2/Bax蛋白比率,降低缺血再灌注后大鼠心肌细胞凋亡指数发挥抗细胞凋亡作用;3.初步证明Cx43可能参与了舒芬太尼预处理对大鼠的心肌保护作用,阿片受体、蛋白激酶C介导了舒芬太尼预处理的心肌保护。更多还原

【Abstract】 Objective①The study was aimed to investigate the protective effect of sufentanil preconditioning against myocardial ischemicreperfusion injury in rats;②To approach the influence of myocyte apoptosis of ischemic-reperfusion rats and the regular effect of Bcl-2 gene and Bax gene after sufentanil preconditioning;③To investigate the effect of naloxone and protein kinase C antagonist on the expression of connextin 43(Cx43) protein and the level of myocardial connextin43 mRNA after sufentanil preconditioning,to test whether the myocardial preservation effect of sufentanil preconditioning is mediated by opioid receptor,portein kinase C and Cx43.Methods The experiment consisted of three parts:①BW of male SD rats was 250-350g.The thoracotomy and left anterior descending coronary artery was ligated.Animals were randomized to divide into six groups(n=8 each).sham-operated rats(group S) were treated identically except the left coronary artery was not tied.Ischemicreperfusion group(group I/R) was treated 30 min of occlusion and 2 h of reperfusion,ischemic preconditioning group(group IPC),ischemic PC was elicited by three 5min occlusion periods interspersed with 5min of reperfusion.Sufentanil preconditioning was with 0.25μg/kg group(group L),1.0μg/kg group(group M) and 5.0μg/kg group(group H) respectively(the total does 0.75,3.0,15.0μg/kg),which was given as three 5min infusions interspersed with a 5min drug-free period before a 30min occlusion period and 2h of reperfusion.The index of cardiac function were recorded at the different time of before and after ischemic-reperfusion.The incidence rate of arrhythmia were observed and graded.The infract size was measured by stained with TTC. Examine the content of MDA and SOD in blood;②According to above-mentioned results,the method of TUNEL and immunohistochemistry was used to detect apoptotic index(AI),the protein expression of Bcl-2 gene and Bax gene in myocardium,which were enforced in group S,group I/R,group IPC and group SPC(sufentanil preconditioning was with 3.0μg/kg group).Mean optical density (OD) value of the positive fields of protein expression was quantitatively examined by image analysis system;③Based on the second part,Naloxone,an antagonist of opioid receptor and chelerythrine,an inhibitor of protein kinase C were given alone(group Nal and group Che) or before sufentanil preconditioning (group Nal+SPC and group Che+SPC).The intracellular distribution of Cx43 in myocardium was observed by light microscope.The expression of connextin43 protein and the level of myocardial connextin 43 mRNA were determined by method of immunohistochemistry and reverse transcriptase polymerase chain reaction(RT-PCR) respectively.Results①There were no significant difference in HR,MAP, LVDP and±dp/dtmax at each group to compred with group S(P＞0.05), but HR,MAP,LVDP and±dp/dtmax were lower in group I/R than that in group S.To compred with the moment before ischemic(T0),MAP,LVDP and +dp/dtmax was significantly decrease at different time during ischemic- reperfusion in group I/R(P＜0.05 or P＜0.01).The +dp/dtmax were lower than basal level(T0) after reperfusion 30min and 120 min in groupL(P＜0.05).LVDP and±dp/dtmax step up gradually after reperfusion 30 min in group IPC and every group in sufentanil preconditioning,and there were not significant difference to compred with basal level(T0) (P＞0.05).The arrhythmia score and IS/AAR in group IPC and in erery group of sufentanil preconditioning were lower than that in group I/R(P＜0.01).MDA was lower obviously in erery group of sufentanil preconditioning than that in group I/R(P＜0.05),and group H was lower than group S.SOD were higher than group I/R,but it were lower than group S(P＜0.05);②AI was very low in group S,but with a more obviously increasing in group I/R(P＜0.01).The cells of apoptosis can be seen in myocardium of group IPC and group SPC,AI of both groups was higher than that in group S,but were decreased significantly as compared with the group I/R(P＜0.01).The protein expression of Bcl-2 gene and Bax gene in myocardium was increased significantly in group I/R,group IPC and group SPC(P＜0.01).The OD values of Bcl-2 gene in group IPC and group SPC was higher than that in group I/R,but the OD values of Bax was reverse.Meanwhile to compared with the group I/R,the ratio of Bcl-2/Bax was much higher in group IPC and group SPC(P＜0.01);③To compared with the group I/R,IPC and SPC were up-regulation the expression of Cx43 protein and the level of myocardial Cx43 mRNA(P＜0.05 or P＜0.01).Naloxone and chelerythrine could reverse the increase of connextin43 protein and it’s mRNA caused by sufentanil preconditioning,so with no significant changes in group Nal+SPC and group Che+SPC.Conclusion①The sufentanil preconditioning can remarkably enhance cardial-protective effect against myocardial ischemic-reperfusion injury in rats.And the effect of sufentanil reduce incidence rate of arrhythmia and myocardial infarct size were the more effective when the dose of management was 3.0 and 15.0μg/kg;②The effectiveness of anti-apoptotic caused by sufentanil precondit -ioning,which was by up-regulation with the protein expression of Bcl-2 gene,and down-regulation with the protein expression of Bax.In addition, it elevated the ratio of Bcl-2/Bax and reduced AI;③It was first demonstration that Cx43 has may participate in the cardial-protection of ischemic preconditioning and sufentanil preconditioning, and the mechanism of sufentanil preconditioning may involve opioid receptor and portein kinase C pathway.更多还原