梗死相关血管晚期再灌注对实验性心肌梗死心肌细胞凋亡的影响

Late Reperfusion of Infarct-Related Coronary Artery on Cardiocyte Apoptosis After Acute Myocardial Infarction in Dogs

目的探讨犬急性心肌梗死后梗死相关血管晚期再灌注对梗死周边缺血区心肌细胞凋亡以及凋亡相关基因bcl-2、bax及其相关蛋白表达的影响。方法健康成年杂交犬28只,全身麻醉下常规开胸暴露冠状动脉后随机分为三组:假手术组(n=8)、急性心肌梗死组(n=10)和晚期再灌注组(n=10)。假手术组仅行左冠状动脉前降支下穿过丝线而不结扎冠状动脉,急性心肌梗死组行左冠状动脉前降支高位永久结扎,晚期再灌注组在高位结扎左冠状动脉前降支6 h后松解结扎线予以6 h的再灌注处理。共有23只犬模型制作成功。各组犬均于术后12 h处死,采集心肌标本。使用脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记法检测心肌细胞凋亡,逆转录聚合酶链反应法检测心肌细胞bcl-2和bax mRNA表达水平,免疫组织化学染色和蛋白印迹法分析bcl-2、bax在心肌细胞中的表达情况。结果晚期再灌注组心肌细胞凋亡指数较急性心肌梗死组明显减少(P<0.05),且两组心肌细胞凋亡指数均高于假手术组(P<0.01)。急性心肌梗死组和晚期再灌注组bcl-2和bax mRNA的表达均明显高于假手术组(P<0.01),且晚期再灌注组bax mRNA的表达明显低于急性心肌梗死组(P<0.05),而bcl-2 mRNA两组间差异无显著性。与假手术组相比,急性心肌梗死组和晚期再灌注组bcl-2蛋白的表达均明显升高(P<0.01),其中在晚期再灌注组的表达略高于急性心肌梗死组,但差异无显著性;晚期再灌注组bax蛋白的表达明显高于假手术组(P<0.01),但明显低于急性心肌梗死组(P<0.05)。结论急性心肌梗死后晚期再灌注可以减少梗死周边缺血区心肌细胞凋亡,其机制可能与心肌细胞bax基因及其相应的蛋白表达减少有关。

Aim To study the effects of late reperfusion on cardiocyte apoptosis and the expression of bel-2 and bax mRNA and related proteins in the risk areas after acute myocardial infarct in dogs. Methods After chest was opened and the coronary artery was exposed, twenty eight dogs were randomly divided into three groups： sham operation group （n=8）, acute myocardial infarction group （n=10）, and late repeffusion group （n=10）. Apart from sham operation group, the left anterior descending arteries were occluded for 12 h in acute myocardial infarction group and 6 h occlusion followed by 6 h of reperfusion in late reperfusion group. The cardiocyte apoptosis was measured by TUNEL assay, the expression of bel-2 and bax gene mRNA by RT-PCR. Immunohistocbemistry and Western blot analysis were used to detect the expression of bel-2 and bax protein. Resuits The apoptoltic index of cardiocyte in late reperfusion group was much higher than that of sham operation group （P〈0.01 ）, but less than that of acute myocardial infarction group （P〈0.05 ）. Compared with sham operation group, the expression of bcl-2 mRNA and protein were significantly increased in acute myocardial infarction group and late reperfusion group （P〈0.01 ）, and there were no significant difference between acute myocardial infarction group and late reperfusion group （P〉0.05 ）. The expression of bax mRNA and its protein in late reperfusion group was much higher than that of sham operation group （P〈0.01）, but lower than that of acute myocardial infarction group （P〈0.05）. Conclusions Late nreperfusion of infarct-related artery can decrease cardiocyte apoptosis in the risk area of acute myocardial infarction, which may be relted to the reduction of the expression of bax mRNA and protein.