转染Akt-1基因对大鼠心肌缺血-再灌注损伤的保护作用研究

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±do／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，Western Blot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。

Study of the Protective Effects of Transgenic Akt-1 Gene After Cardiac Ischemia-reperfusion Injury in Rats

Objective To investigate the protective effects of transgenic Akt-1 gene on rats after ischemia-reperfusion injury in vivo. Methods 40 adult male SD rats were divided randomly into five groups with 8 rats in each group： sham operation group（S）, ischemia/reperfusion group（I-R）, Akt-1 gene group（Gene）, Vechicle Control group（VC）, Block group（AB）. The rats in Gene group were injected 30μ1 Lipofectamine 2000 solution including Akt-1 gene to the myocardium 48 hours before ischemia while those in S group and I-R group were injected PBS of the same volume. In VC group, myocardium was injected Lipofectamine 2000 of the same volume. 30/xl Lipofectamine 2000 and gene complexes with LY29dO02 was injected in AB group. All rats were intubated to left ventricle and the changes of HR, LVSP, LVEDP, ＋ dp/dtmax and -dp/dtmax were recorded.The apoptotic cells were assessed by TUNEL staining and apoptosis index （AI） was obtained, and the myocardial infarction area was evaluated by TIC dyeing. The changes of Bcl-2, Bax in myocardial cells were observed by immunohistochemistry. The expression of Akt-1 and Caspase-3 was determined by Western Blot . Results 1. The left ventricular function （LVF） of S group was higher than other groups （P 〈 0.05）. LVF of I-R group, VC group and AB group were lower than Gene group （P 〈 0. 05）. 2. Compared with those in S group, apoptosis index（AI） and myocardial infarction area increased significantly in other groups （P 〈0. 05）. The M and myocardial infarction area in Gene group were significantly lower than those in I-R group, VC group and AB group （P 〈0.05 ）. 3. In S group, the level of protein expression Akt-1 and Caspase-3 is the lowest, the protein expression of Akt-1 in Gene group is higher than I-R group, VC group and AB group, while the protein expression of Caspase-3 is on the contrary. 4. Compared with S group, the expression of Bcl-2 and Bax in other groups increased（ P 〈 0. 05） ; the expression of Bcl-2 in Gene group is higher