【作者】 刘勇；

【导师】 孙宗全；

【作者基本信息】 华中科技大学 ， 心血管外科学， 2010， 博士

【摘要】 目的：研究心肌缺血期短暂肢体缺血处理(remote perconditioning, RP)对大鼠心肌缺血再灌注(I／R)损伤的保护作用。方法：健康雄性Wistar大鼠经胸骨下段切口暴露心脏,打活结的方式结扎或旷置冠状动脉左前降支(LAD)后,经心尖置测压管于左心室,建立在体心肌I/R损伤模型,随机分为7组。(1)假手术组(Sham组,n=8)：经冠状动脉左前降支(LAD)下穿线后,旷置；(2)缺血／再灌注组(IR组,n=12)：心脏LAD实施30min缺血／180min再灌注处理；(3)心肌缺血期短暂肢体缺血处理组(RP组,n=10)：心脏LAD实施30min缺血／180min再灌注处理；在实施心肌缺血期间对双下肢实施3次5min缺血／5min再灌注;(4)短暂肢体缺血预处理组(RIPC组,n=11)：心肌缺血前先对双下肢实施3次5min缺血／5min再灌注,而后对心脏LAD实施30min缺血／180min再灌注处理；(5)短暂肢体缺血后处理组(RI-PostC组,n=11)：对心脏LAD实施30min缺血／180min再灌注处理；再灌注开始的同时对双下肢实施3次5min缺血／5min再灌注；(6)格列苯脲组(GL组,n=11)：心脏LAD实施30min缺血／180min再灌注处理；在实施心肌缺血期间对双下肢实施3次5min缺血／5min再灌注；同时腹腔注射线粒体ATP敏感性钾通道(mKATP)抑制剂格列苯脲5mg/kg;(7)环孢霉素A组(CS组,n=10)：心脏LAD实施30min缺血／180min再灌注处理；心肌缺血期间经心脏测压管注射线粒体通透性转换孔(mPTP)抑制剂环孢霉素A (CsA) 10mg/kg。从再灌注性心律失常(reperfusion arrhythmia, RA)、心肌舒缩能力、心肌损伤标志物、心梗范围、心肌细胞凋亡相关蛋白的表达等方面评价RP对心肌IRI的保护作用。计算缺血期心律失常(ischemia arrhythmia, IA)及RA评分、左心室发展压(LVDP)、左心室压上升／下降最大速率(±dp/dtmax)及其恢复率；ABC夹心ELISA法检测血清肌酸激酶(CK)活性和心肌肌钙蛋白T(c-TNT)、HE染色观察心肌形态学改变,TTC染色法测定心肌梗死面积,免疫组化法检测心肌细胞凋亡相关蛋白Bcl-2、Bax的表达,免疫印迹法(western blot)检测心肌凋亡蛋白caspase-3的表达。结果：1.动物模型的评价IR组与Sham组比较,心肌缺血期ST段显著抬高(P<0.01),缺血期及再灌注期心律失常评分显著增加(P<0.05),左心室收缩峰压(LVSPmax),±dp/dtmax在再灌注起点时下降,差异均具有统计学意义(P<0.05),c-TNT显著升高(P<0.05)。2.RP对大鼠心肌I／R损伤所致RA、心脏舒缩功能及心梗范围的影响再灌注早期RP组RA评分为(1.50±0.97),与R组(2.33±0.71)差异有统计学意义(P=0.049<0.05)；RP组的LVSPmax、±dp/dtmax均介于Sham组与IR组之间,但与R组的差异无统计学意义。三组间CK差异无统计学意义,关于c-TNT, IR组与Sham组的差异具统计学意义(P=0.021),但RP组与IR组、sham组间差异均无统计学意义。三组关于左室心肌危险区域(缺血范围)无显著差异,RP组心梗范围显著小于IR组(P<0.01)。3.RP对大鼠心肌I／R损伤所致细胞凋亡的影响比较R组与RP组的Bcl-2、Bax的表达结果发现,与R组相比,RP组Bax蛋白表达阳性心肌细胞百分比明显降低(P<0.05),而Bcl-2蛋白表达阳性百分比无显著性差异(P>0.05),Bcl-2/Bax比值明显升高(P<0.05)。RP组caspase-3的表达与IR组间无显著性差异(P>0.05)。4.RP、RIPC、RI-PostC模式对大鼠心肌I／R损伤影响的比较再灌注早期RP组RA评分与IR组之间差异显著(P<0.05),但RIPC组、RI-PostC组与IR组、RP间差异均无统计学意义。RP组与R组间血流动力学数据无显著性差异。RIPC组在再灌注30 min时LVDPmin高于R组,RI-PostC组-dp/dtmax再灌注0 min时显著低于RP组(P<0.05),其余数值差异无统计学意义。四组间CK与c-TNT均无显著差异。RP、RIPC、RI-PostC组心梗范围显著低于R组(P<0.01),RP、RIPC、RI-PostC三组间无显著差异。RP组、RIPC组、RI-PostC组Bcl-2表达均高于IR组,RI-PostC组表达高于RP组与R组,RIPC组表达高于IR组(P<0.05)。RP组、RIPC组、RI-PostC组关于Bax表达相当,RP组、RIPC组显著低于R组(P<0.05)。R组Bcl-2/Bax阳性单位的比值低于RP、RI-PostC、RIPC三组,与RP组差异有统计学意义(P<0.05)。RP组、RIPC组、RI-PostC组三组间关于Bcl-2、Bax、Bcl-2/Bax差异无统计学意义。四组间caspase-3蛋白的表达差异无统计学意义。5.应用格列苯脲干预RP处理对大鼠心肌I／R损伤的影响GL组再灌注早期RA评分低于IR组,差异具有统计学意义(P<0.05),但与RP组无显著差异。在再灌注0 min时,GL组LVSPmax及±dp/dtmaX高于R组(P<0.05)。GL组与RP组间无显著差异。GL组c-TNT表达低于R组及RP组(P<0.05)。GL组心梗范围介于RP、R组之间,与RP组差异具有统计学意义(P<0.05),与R组差异无统计学意义。RP组、GL组关于Bcl-2表达相当,均高于IR组,GL组与R组间具有显著性差异(P<0.05)。GL组关于Bax表达介于RP、IR组之间,但差异无统计学意义。三组间Bcl-2/Bax阳性单位比值的差异缺乏统计学意义。GL组caspase-3的表达显著高于IR组和RP组,具有统计学意义(P<0.05)。6.心肌缺血期应用CsA对大鼠心肌I／R损伤的影响CS组再灌注早期RA评分低于R组,差异具有统计学意义(P<0.05),但与RP组差异无统计学意义。CS组再灌注0、60 min, LVDPmin均高于IR组；再灌注120 min时,LVSPmax低于IR组；再灌注30-180 min的±dp/dtmax恢复率均低于IR组。再灌注60 min时-dp/dtmax,再灌注120min时LVSPmax,再灌注180 min时dp/dtmax低于RP组；再灌注30-180 min,-dp/dtmax恢复率及30-60 min dp/dtmax恢复率均低于RP组；再灌注120min, LVDP低于RP组(P<0.05)。CS组c-TNT表达低于R组及RP组,但无统计学意义。CS组心梗范围介于RP、IR组之间,显著小于R组(P<0.05)。RP、CS组关于Bcl-2表达相当,均高于IR组,但差异无统计学意义。CS组Bax表达介于IR, RP组之间,但与两组差异无统计学意义。关于Bcl-2/Bax阳性单位的比值的排序为：R组<CS组<RP组,CS组显著低于RP组(P<0.05)。CS组caspase-3的表达高于IR组(P<0.05)组,但与RP组差异无统计学意义。结论：1.经胸骨下段切口建立大鼠心肌缺血／再灌注模型简便易行,监测结果稳定可靠。2.RP能减少大鼠心肌I／R损伤,表现为减轻再灌注性心律失常,缩小心肌梗死面积,改善心肌形态学损伤,保护左心室收缩、舒张功能。RP可能通过减少促凋亡蛋白Bax的表达来发挥对IRI诱发的心肌细胞凋亡的抑制作用。3.肢体短暂缺血模式进行的RIPC、RP、RI-Postc三种模式均具有心肌保护作用,表现为减小心肌梗死范围,抑制心肌细胞凋亡,且保护能力相当。4.腹腔注射格列苯脲可以减轻心肌缺血性心律失常,也部分抵消了RP的心肌保护作用。心腔注射CsA可以模拟部分RP的心肌保护作用,但其对心肌的隐匿副损伤抵消了其它的有益作用。线粒体ATP敏感性钾通道(mKATP)和线粒体通透性转换孔(mPTP)仍是RP诱导内源性心肌保护的最重要效应器。更多还原

【Abstract】 Objective:To study the protection induced by transient limb ischemic conditioning during myocardial ischemic period against myocardial ischemia reperfusion (I/R) injury of rats.Method:Healthy male Wistar rats were administered. The heart was exposed through lower sternal incision and the left anterior descending coronary artery was ligated by snipknot to establish the ischemia/reperfusion model. A catheter was put into the left ventricle from the apex of heart to trace the pressure of left ventricle and the pressure change index while electrocardiogram was monitored. Rats were assigned randomly into seven groups. (1)Sham group (n=8):rats were threaded a silk suture under the left coronary artery anterior descending (LAD) and laid up through out the experiment. (2)IR group (n=12):rats were subjected to 30 min LAD occlusion followed by 180 min reperfusion. (3) RP group (transient limb ischemic conditioning during myocardial ischemia period, remote preconditioning group) (n=10):rats were subjected to three episodes of 5 min double hind limbs occlusion followed by 5 min reperfusion during 30 min myocardial ischemia period which follower by 180 min reperfusion. (4) RIPC group (remote ischemia preconditioning group) (n=11):rats were subjected to three episodes of 5 min double hind limbs occlusion followed by 5 min reperfusion before 30 min myocardial ischemia period which follower by 180 min reperfusion. (5) RI-PostC group (remote ischemia post-conditioning group) (n=11):rats were subjected to three episodes of 5 min double hind limbs occlusion followed by 5 min reperfusion at the very beginning of reperfusion during 30 min myocardial ischemia and 180 min reperfusion period. (6)GL group (Glibenclamide group) (n=11):rats were subjected to three episodes of 5 min double hind limbs occlusion followed by 5 min reperfusion during 30 min myocardial ischemia period while glibenclamide was injected intraperitoneally dosed with 5 mg/kg which also follower by 180 min reperfusion. (7)CS group (Cyclosporin A group) (n=10):rats were subjected to 30 min LAD occlusion followed by 180 min reperfusion while CsA was injected into the left ventricle from the catheter dosed with 10 mg/kg.The myocardial protection induced by RP against IRI was evaluated by reperfusion arrhythmia (RA), cardiac contractility, marker of myocardial injury, infarct size, cardiocyte death, expression of apopto sis-relevant proteins. The scores of ischemia arrhythmia and reperfusion arrhythmia, the left ventricular developing pressure, the maximal change index of LV (±dp/dtmax) and recovery percentage of maximal change index were calculated. Serum CK (creatine kinase) and c-TNT (cardiac troponin T) were tested by ABC ELISA. Myocardial definite change of tissue morphology was inspected by HE staining. Myocardial infarct size was measured by TTC staining technique. The expression of apoptosis-relevant proteins, Bcl-2 and Bax, were tested by histochemistry immunity method, and caspase-3 was tested by western blot.Results:1. Evaluation of animal model.Compare IR group to Sham group, the ST segment of ischemia period was significant elevated(P<0.01), the scores of ischemia and reperfusion arrhythmia were significant increased(P<0.05), the maximum systolic pressure of left ventricular and the idp/dtmax at the reperfusion starting point were significantly decreased, and the serum c-TNT was significantly higher (P<0.05).2. Influence on RA, cardiac contractility and infarct size of remote perconditioning against rats’ischemia reperfusion injury.The RA score of early reperfusion phase, change index and maximal pressure of left ventricle at end point of RP group all fell in between IR group and sham group. The arrhythmic score of RP group was (1.50±0.97) and the score of IR group was (2.33±0.71). The difference between them was statistically significant (P=0.049). There was no statistically significant difference on change index and maximal pressure of left ventricle between IR group and RP group. Difference of CK among three groups was not statistically significant. While the c-TNT of IR group was significant higher than that of sham group (P=0.021), there was no statistically significant difference between RP and IR group or between RP and sham group. There was no difference of ischemia size among three groups, while infarct sizeof RP group was significant smaller than that of IR group (P<0.01).3. Influence on cardiocyte apoptosis against rats’myocardial IRI.Compared to IR group, the percentage of cardiocyte of RP group which expressed Bax was statistically significantly lower (P<0.05), while the percentage of Bcl-2 was not (P>0.05). The rate of Bcl-2/Bax was statistically significantly higher (P<0.05). There was no significant difference on expression of caspase-3 between RP and IR group (P>0.05).4. Comparison among RP, RIPC and R1-PostC induced protection against rats’ myocardial IRI.RA score of RP group was smaller than IR group (P<0.05) but it of RIPC and Rl-PostC group had no statistically significant difference compared to both RP and IR groups. There were no statistically significant difference of hemodynamics data between RP group and IR group. Compared to IR group, LVDPmin of RIPC group were higher at reperfusion 30 min (P<0.05) and-dp/dtmax of RI-PostC group was lower at reperfusion 0 min (P<0.05). Other data difference was not statistically significant. Difference of serum CK and c-TNT among four groups was not statistically significant. There was no difference of ischemia size among four groups, while the infarct size of RP, RIPC and RI-PostC group was statistically significantly smaller than IR group (P<0.01). The infarct size percentage of RP group was (15.27±5.19), it of RIPC group was (14.53±3.45), and it of RI-PostC group was (19.84±5.85), while the size percentage of IR group was (34.47±7.13). There was no difference of infarct size among three conditioned groups.Expression of Bcl-2 in three groups was higher than it of IR group. Bcl-2 of RI-PostC group was statistically significant higher than it of RP and IR groups while that of RIPC group was higher than that of IR group (P<0.05). Expression of Bax in three groups was similar and those of RP and RIPC group were statistically significant lower than it of IR group (P<0.05). The Bcl-2/Bax positive unit rate of IR group was lower than RP, RIPC and R1-PostC group. The difference between IR and RP group was statistically significant (P<0.05). There was no significant difference on Bcl-2, Bax and Bcl-2/Bax among three conditioned groups. There was also no significant difference on caspase-3 among these four groups.5. Impact of glibenclamide on remote perconditioning induced myocardial protection.RA scores (P<0.05) of GL group was lower than IR group. The LVSPmax and±dp/dtmax were higher than IR group (P<0.05) at reperfusion 0 min. But there were no significant difference of arrhythmia scores and cardiac contractility compared to RP group. Serum c-TNT of GL group was lower than RP and IR group (P<0.05). The infarct size of GL group fell in between RP group and IR group, the difference compared to RP group was statistically significant (P<0.05) while it compared to IR group was not.Expression of Bcl-2 in RP group and GL group were higher than IR group and there was statistically significant difference between GL and IR group. Expression of Bax in GL group fell in between RP group and IR group, while no statistically significant difference was found compared to RP and IR group. The difference of Bcl-2/Bax positive unit rate wasn’t significant. Expression of caspase-3 in GL group was higher than both IR and RP group (P<0.05).6. Influence of CsA applied during myocardial ischemia period against rats’ myocardial IRI.RA scores (P<0.05) of CS group was lower than IR group, while no significant difference was found compared to RP group. Compared to IR group, LVDPmin was higher at reperfusion 0 and 60 min; LVSPmax was lower at reperfusion 120 min; recovery percentage of±dp/dtmax was lower from reperfusion 30 min to 180 min(P<0.05).Compared to RP group, LVSPmax,-dp/dtmax at reperfusion 60 min, LVSPmax at 120 min and±dp/dtmax at 180 min of CS group were lower; recovery percentage of-dp/dtmax from 30 to 180 min and dp/dtmax from 30 to 60 min were lower; LVDP at 60 min was also lower than RP group (P<0.05).Serum c-TNT of CS group was lower than RP and IR group but not statistically significant. The infarct size of CS group fell in between RP group and IR group, the difference between CS and IR group was statistically significant (P<0.05). Expression of Bcl-2 in RP group and CS group were higher than IR group but there was no statistically significant difference. Expression of Bax in CS group fell in between RP group and IR group, while no statistically significant difference was found. The sequence of Bcl-2/Bax positive unit was IR group, CS group and RP group. The difference compared to RP group was significant (P<0.05). Expression of caspase-3 in CS group was higher than IR group (P<0.05), but had no significant difference with RP group.Conclusions:1.The method of establish rat myocardial ischemia/reperfusion model througu lower sternal incision is simple and convenient, and the experiment results are reliable.2.RP method can reduce rats’myocardial IRI. It was found to alleviate RA, decrease infarct size, improve myocardial morphological lesion, and protect left ventricle contractility. It maybe the mechanism that RP inhibits IRI induced cardiocyte apoptosis by decreasi ng expression of Bax, the pro-apoptotc protein.3.The three methods of RP, RIPC and RI-PostC executed by transient limb ischemia conditioning all have equal myocardial protection ability against IRI, displayed as decreased infarct size, inhibited cardiocyte apoptosis. RP method is even better on alleviating RA.4.Glibenclamide injected intraperitoneally can alleviate ischemia arrhythmia score and partly counteract myocardial protection induced by RP. CsA injected into heart chamber can partly simulate RP method and induce some protection. But its hiding injury can abolish its benign effect. The most important effecters of RP induced endogenous myocardial protection is mKATP and mPTP.更多还原