心肌细胞内Livin蛋白过表达对大鼠心肌缺血再灌注中细胞凋亡的影响

Effects of Livin Over-Expression on Cardiomyocyte Apoptosis During Myocardial Ischemia Reperfusion Injury in Rats

目的:观察大鼠心肌缺血再灌注损伤过程中心肌细胞内黑色素瘤凋亡蛋白抑制剂(Livin)蛋白表达变化及Livin蛋白过表达对心肌细胞凋亡的影响。方法:健康SD大鼠80只,随机分为假手术组、缺血再灌注组、注射表达Livin蛋白的逆转录病毒载体组(Livin组)和空载体组,其中Livin组和空载体组分别向开胸心肌内注射表达Livin的逆转录病毒载体或空载体,24h后行缺血再灌注过程,以实时荧光定量逆转录多聚酶链反应(RT-PCR)测定心肌组织内Livin和天冬氨酸特异的半胱氨酸蛋白酶-3(Caspase-3)信使核糖核酸(mRNA)的表达,免疫组织化学方法检测Caspase-3的表达,以原位缺口末端标记(TUNEL)法测定凋亡心肌细胞。结果:①实时荧光定量RT-PCR扩增结果LivinmRNA相对表达水平:与假手术组比较Livin组显著增高(P<0.01);与缺血再灌注组比较Livin组升高(P<0.01)。Caspaes-3mRNA相对表达水平:与假手术组相比缺血再灌注组、Livin组、空载体组均增高(P<0.01);与缺血再灌注组比较Livin组降低,差异有统计学意义(P<0.01)。②免疫组化显示:与假手术组比较缺血再灌注组、Livin组、空载体组缺血再灌注过程中Caspase-3的表达明显增加(P<0.05);与缺血再灌注组比较Livin组表达减少(P<0.05),差异均有统计学意义。③TUNEL法检测:与缺血再灌注组比较假手术组、Livin组凋亡细胞数明显减少,差异均有统计学意义(P<0.05)。结论:心肌Livin蛋白过表达可以抑制缺血再灌注心肌Caspase-3表达,抑制缺血再灌注心肌细胞凋亡的发生。

Objective ： To study the changes of Livin and caspase-3 mRNA expression and Livin over expression on myocardium, and the effect of cardiomyocyte apoptosis during myocardial ischemia reperfusion （IR） injury in rats. Methods： A total of 80 healthy SD rats were divided into d groups, n = 20 in each group. Sham operation group （S group） ; IR group, the rats were subjected to 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion; Livin group, the rats were transected with retrovirus vector expressing Livin protein by intra-myocardial injection 24 hours before left coronary artery occlusion; and Empty-vector control group （Control group）. Livin mRNA expression, Caspase-3 mRNA expres- sion and Caspase-3 protein expression were detected by real time RT-PCR and immunohistochemistry. Cardiomyocyte apoptosis was determined by TDT-mediated dUTP nick end labeling （TUNEL） method. Results： （1） Compared with S group and IR group, real time RT-PCR displayed that Livin mRNA expression was significantly increased after the transfection in Livin group （P 〈 0. 01 respectively）. Compared with S group, Caspase-3 mRNA expres- sion significantly increased in IR group, Livin group and Control group （P 〈 0. 01 respectively）, while compared with IR group, Caspase-3 mRNA expression was decreased in Livin group （P 〈 0. 01 ）. （2） According to immunohistochemistry, compared with S group, Caspase-3 protein expression significantly increased in IR group, Livin group and Control group during isehemia reperfusion（P 〈 0. 05 respectively）, while compared with IR group, Caspase-3 expression decreased in Livin group （P 〈 0. 05）. （3） As indicated by TUNEL method, compared with IR group, the cardiomyocyte apoptosis index was much lower in S group and Livin group （P 〈0. 05 respectively）. Conclusion ： The over expression of Livin could suppress Caspase-3 expression and inhibit the cardiomyocyte apoptosis during myocardial ischemia reperfusion injury in rats.