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目的:研究Akt1基因对缺血再灌注(I/R)损伤心肌细胞的保护作用,并探讨Akt1基因对心肌I/R损伤时线粒体通透性转换(MPT)功能的影响。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120min,建立大鼠在体心肌I/R损伤模型。40只雄性SD大鼠随机分为5组,每组8只:假手术对照组(control组)、心肌缺血/再灌注组(I/R组)、转Akt1基因+I/R组(Ad-gene组)、空载体+I/R组(Ad-blank组)、转Akt1基因+I/R+Akt抑制剂组(Ad-inhibitor组)。Ad-gene组术前48h开胸心肌内直接分点注射脂质体Akt1质粒复合物,control组和I/R组分别注射同等体积磷酸盐缓冲液(PBS),Ad-blank组注射等体积脂质体,Ad-inhibitor组注射等体积脂质体Akt1质粒LY294002混合物。观察转染Akt1基因对大鼠I/R心脏乳酸脱氢酶(LDH)和肌酸激酶(CK)释放、心肌细胞凋亡、Akt1表达、线粒体和胞浆内细胞色素C(Cyc-C)表达以及MPT的影响。结果:Control组可见Akt1表达,但明显低于其余各组;与control组相比较,其余各组心肌细胞凋亡指数(AI)、LDH和CK均增高;与control组相比较,其余各组Cyc-C较胞浆表达增加而线粒体内表达减少。Ad-gene组Akt1蛋白表达较control组、I/R组、Ad-blank组及Ad-inhibitor组均增加;Ad-gene组AI、LDH和CK较I/R组、Ad-blank组及Ad-inhibitor组均显著减少,但仍高于control组;Ad-gene组Cyc-C较I/R组、Ad-blank组以及Ad-inhibitor组胞浆内表达减少而线粒体内表达增加;Ad-gene组较IR组、Ad-blank组和Ad-inhibitor组在540nm处吸光度值增高,但明显低于control组。I/R组、Ad-blank组以及Ad-inhibitor组Akt1、Cyc-C、AI、LDH和CK以及在540nm处吸光度值比较均无显著差异。结论:转染Akt1基因对I/R损伤心肌细胞具有保护作用,该作用可能与Akt1抑制I/R诱导的心肌线粒体膜通透性转换,从而保护线粒体的功能有关。
AIM: To investigate the protective effect of Akt1 gene on myocardial cells injured by ischemia / reperfusion (I / R) and the effect of Akt1 gene on mitochondrial permeability transition (MPT) function during myocardial I / R injury. Methods: The ligation of the left anterior descending coronary artery was performed in rats. Ligation of the anterior descending coronary artery was performed for 30 min and reperfusion for 120 min. Forty male Sprague-Dawley rats were randomly divided into 5 groups: control group, I / R group, Akt1 + I / R group -gene group), empty vector + I / R group (Ad-blank group) and Akt1 gene + I / R + Akt inhibitor group (Ad-inhibitor group). In the Ad-gene group, the liposome Akt1 plasmid complex was directly injected into the thoracotomy myocardium 48 hours before the operation. The control group and the I / R group were injected with the same volume of phosphate buffered saline (PBS) Plastid, Ad-inhibitor group were injected with an equal volume of liposomes Akt1 plasmid LY294002 mixture. The effect of Akt1 gene transfection on the release of LDH and CK, the expression of Akt1, the expression of mitochondria and cytochrome C (Cyc-C) in cardiomyocytes of rats with I / R heart was observed As well as the impact of MPT. Results: Compared with the control group, the expression of Akt1 in the control group was significantly lower than that in the control group. Compared with the control group, the apoptotic index (AI), LDH and CK in the other groups were all increased. Compared with the control group, C increased compared with cytoplasm and decreased in mitochondria. Ad-gene group Akt1 protein expression than control group, I / R group, Ad-blank group and Ad-inhibitor group were increased; Ad-gene group AI, LDH and CK than I / R group, Ad-blank group and Ad- The expression of Cyc-C in Ad-gene group was lower than that in I / R group, Ad-blank group and Ad-inhibitor group, but increased in mitochondria. Ad-gene group Compared with IR group, Ad-blank group and Ad-inhibitor group increased the absorbance value at 540 nm, but significantly lower than the control group. Akt1, Cyc-C, AI, LDH and CK in I / R group, Ad-blank group and Ad-inhibitor group did not show significant difference compared with absorbance value at 540 nm. CONCLUSION: Transfection of Akt1 gene has a protective effect on I / R injured myocardial cells, which may be related to Akt1 inhibiting I / R-induced permeability transition of mitochondrial membrane and protecting mitochondrial function.