重组人红细胞生成素对心肌梗死治疗作用的实验研究

An experimental study of recombinant human erythropoietin on the treatment of acute myocardial infarction in rats

目的观察重组人红细胞生成素(rHu-EPO)对大鼠心肌梗死的治疗作用并探讨其机制。方法体外培养出生3 d 内 SD 大鼠心肌细胞,缺氧培养和 H_2O_2模拟氧化应激反应诱导凋亡,培养体系中加入 rHu-EPO,荧光染色观察凋亡率的变化。32只成年 SD 大鼠分空白组8只、心肌梗死组12只和治疗组12只;空白组开胸但不结扎冠脉,心肌梗死组和治疗组结扎左冠前降支制备心肌梗死的模型,治疗组每天腹腔注射5000 IU/kg rHu-EPO 共7 d,14 d 后所有大鼠检测血流动力学指标,心脏切片原位末端标记(TUNEL)检测凋亡,免疫组化检测 Bcl-2、Bax 表达。结果 rHu-EPO 使心肌细胞缺氧诱导的凋亡率由47.92%±5.87%下降为28.21%±4.35%;氧化应激反应所诱导的凋亡率由60.42%±5.54%下降为43.40%±5.39%;腹腔注射 rHu-EPO 使得心肌梗死后大鼠心功能保存较好,动脉收缩压、平均动脉压、左心室收缩压、±dp/dt_(max)都有所提高,同时,左心室舒张压、左心室舒张末期压下降;细胞凋亡率由1.65%±0.50%减少为0.84%±0.30%;Bcl-2蛋白表达率由0.96%±0.37%增加为1.43%±0.28%;Bax 蛋白表达率由1.34%±0.47%减少为0.68%±0.30%。结论rHu-EPO 可抑制心肌细胞凋亡、保存心功能,可能是通过下调 Bax 表达和上调 bcl-2表达实现的。

Objective To observe the treatment and it＇s mechanisms of rHu-EPO on acute myocardial infarction of SD rats in vitro and vivo. Methods Cardiomyocytes were isolated from neonatal Sprague-Dawley rats. Hypoxia condition and oxidative stress were used to induce apoptosis, rHu-EPO was added to the culture system. Apoptosis was assessed by using Hoechst 33258 dyeing. Apoptosis index （AI） was then calculated. Thirty two rats were divided into three groups including sham operation group （Sham）, acute myocardial infarction group （MI） and treated group （MI ＋ EPO）. Acute myocardial infarction model was made by ligating the anterior descending coronary artery, rHu-EPO was administered i.p. in MI ＋ EPO group at the dose of 5000 units/kg of body weight immediately after the ligation and the next six days. At the fourteenth day all animals underwent hemodynamic measurements and then executed, the samples were examined with hematoxylin and eosin （HE） stain, immunohistochemistry technique （ Bcl-2, Bax） and TdT- mediated dUTP nick end labeling （TUNEL） dyeing. Results rHu-EPO significantly down-regulated the apoptosis of cardiomyocytes which underwent hypoxia or oxidative stress. In vivo experiment rHu-EPO protected the hemodynamic function of the rats from myocardial infarction and down-regulated the ratio of the positive cells for TUNEL and Bax. The ratio of the positive cells for Bcl-2 was up-regulated by rHu-EPO. Conclusion These findings suggested rHu-EPO improve myocardial infarction by attenuating apoptosis. Potential mechanism is to up-regulated Bcl-2 expression and down-regulated Bax expression.