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目的通过观察转染Akt-1基因对大鼠心肌缺血-再灌注(I-R)损伤心脏功能及细胞凋亡相关蛋白的影响,探讨Akt-1基因对心肌I-R损伤保护作用的机制。方法使用结扎/松解左冠状动脉前降支法,结扎冠状动脉前降支30min,再灌注2h,建立大鼠在体心肌I-R模型。40只雄性SD大鼠随机分为五组:假手术组(S组),缺血-再灌注组(I-R组),转染Akt-1基因组(Gene组),空载体组(VC组),抑制剂组(AB组),每组8只。Gene组术前48h开胸心肌内直接分点注射脂质体Akt-1质粒复合物,S组和I-R组分别注射同等体积PBS,VC组注射等体积脂质体,AB组注射等体积脂质体Akt-1质粒LY294002混合物。所有大鼠经颈动脉插管至左心室记录HR、LVSP、LVEDP和±dp/dtmax变化,TTC染色法测定心肌梗死范围,TUNEL法原位标记凋亡心肌细胞,WesternBlot测定Akt-1、Casepase-3蛋白表达,免疫组化检测Bcl-2、Bax表达。结果1.Gene组较I-R组、VC组和AB组左室心功能(HR、LVSP、LVEDP、±dp/dtmax)改善(P<0.05)。2.Gene组凋亡指数(AI)及心肌梗死范围较I-R组、VC组及AB组均显著减少(P<0.05),但仍高于S组(P<0.05);S组细胞凋亡阳性细胞几无表达,心肌细胞凋亡指数(AI)明显低于其余各组(P<0.05)。3.各组均可见Akt-1、Casepase-3蛋白表达,但S组中蛋白较其余各组减少(P<0.05);Gene组Akt-1蛋白表达较I-R组、VC组及AB组均增加(P<0.05);Gene组Casepase-3蛋白表达则较I-R组、VC组及AB组减少。4.S组Bcl-2和Bax表达较其余各组减少(P<0.05);Gene组较I-R组、VC组及AB组Bcl-2表达上调而Bax表达下调(P<0.05)。结论Akt-1基因转染对I-R心肌具有保护作用,该作用可能与促进Bcl-2表达,抑制Bax和Casepase-3表达从而抑制凋亡的发生有关。
Objective To observe the effect of Akt-1 gene on cardiac function and apoptosis-related protein during myocardial ischemia-reperfusion (I-R) injury in rats and to explore the mechanism of the protective effect of Akt-1 gene on myocardial I-R injury. Methods The ligation of the left anterior descending coronary artery (LADC) was performed by ligating and releasing the anterior descending coronary artery for 30 minutes and reperfusion for 2 hours. Forty male SD rats were randomly divided into five groups: Sham operation group (S group), ischemia-reperfusion group (IR group), Akt-1 gene transfected group (Gene group), empty vector group Inhibitor group (AB group), each group of eight. In the Gene group, the liposome Akt-1 plasmid complex was directly injected into the myocardium 48 hours after thoracotomy, and the same volume of PBS was injected into the S and IR groups. The same volume of liposomes were injected into the VC group. Body Akt-1 plasmid LY294002 mixture. The changes of HR, LVSP, LVEDP and ± dp / dtmax were recorded by carotid artery catheterization in the left ventricle. The extent of myocardial infarction was measured by TTC staining. Apoptotic cardiomyocytes were labeled by TUNEL method. Akt-1 and Casepase- 3 protein expression, immunohistochemical detection of Bcl-2, Bax expression. Left ventricular function (HR, LVSP, LVEDP, ± dp / dtmax) was significantly improved in Gene group compared with I-R group, VC group and AB group (P <0.05). The apoptosis index (AI) and myocardial infarct size in Gene group were significantly lower than those in IR group, VC group and AB group (P <0.05), but still higher than those in S group (P <0.05) The number of cells did not express, and the apoptosis index (AI) of myocardial cells was significantly lower than the other groups (P <0.05). The expression of Akt-1 and Casepase-3 in each group were significantly lower than those in other groups (P <0.05); Akt-1 protein expression in Gene group was higher than that in IR group, VC group and AB group (P <0.05). The expression of Casepase-3 in Gene group was lower than that in IR group, VC group and AB group. The expression of Bcl-2 and Bax in S group was lower than that in other groups (P <0.05). The Bcl-2 expression in Gene group was significantly higher than that in I-R group, VC group and AB group (P <0.05). Conclusion Transfection of Akt-1 gene has a protective effect on I-R myocardium, which may be related to the promotion of Bcl-2 expression, inhibition of Bax and Casepase-3 expression and inhibition of apoptosis.