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目的探讨miR-221在急性心肌梗死(AMI)大鼠中的表达及其作用。方法在45只SD清洁级雄性大鼠中随机抽取35只通过冠状动脉左前降支结扎大鼠建立AMI模型,将其中建模成功的30只大鼠随机分为AMI组、对照组及抑制组,每组各10只;另抽取未建模的10只大鼠设立假手术组,采用冠状动脉左前降支穿线不结扎。其中对照组用携带绿色荧光蛋白(GFP)的慢病毒空载体miR-221阴性对照以4×10~7病毒数量进行心肌组织局部注射转染,抑制组用携带miR-221 inhibitor慢病毒以4×10~7病毒数量进行心肌组织局部注射转染,AMI组和假手术组每天给予等量生理盐水,隔日1次连续2周;记录大鼠心脏功能。采用原位末端凋亡法(TUNEL)检测心肌凋亡指数(AI),RT-PCR法检测miR-221表达,Western blot法检测Bax、Bcl-2、PI3K和p-AKT蛋白表达。结果与假手术组大鼠miR-221表达量(0.18±0.02)比较,AMI组(1.16±0.12)和对照组(1.18±0.12)大鼠的miR-221表达量均升高(均P<0.01),抑制组大鼠miR-221表达量(0.30±0.03)均低于AMI组和对照组大鼠的miR-221表达量(均P<0.01);与假手术组大鼠Bax(0.13±0.01)、Bcl-2(0.53±0.05)、PI3K(0.45±0.04)和p-AKT(0.87±0.09)蛋白表达量比较,AMI组和对照组大鼠的Bax蛋白表达量均上调(均P<0.01),Bcl-2、PI3K和p-AKT蛋白表达量均下调(均P<0.01);抑制组大鼠较AMI组和对照组大鼠Bax表达量均下调(P<0.01),Bcl-2、PI3K和p-AKT蛋白表达量均上调(P<0.01)。结论 miR-221在AMI大鼠心肌组织中高表达,下调miR-221表达可通过激活PI3K/AKT信号通路调控下游蛋白Bax及Bcl-2的表达抑制AMI大鼠心肌细胞凋亡。
Objective To investigate the expression and its role of miR-221 in acute myocardial infarction (AMI) rats. Methods Thirty-five male Sprague-Dawley rats were randomized to take an acute myocardial infarction (AMI) model by ligating their left anterior descending coronary artery. 30 rats were randomly divided into AMI group, control group and control group. Each group of 10; the other 10 non-modeling rats were set up sham operation group, using the left anterior descending coronary artery threading not ligation. The control group was transfected with 4 × 10 ~ 7 virus by local injection with the lentiviral empty vector miR-221 negative control carrying green fluorescent protein (GFP) The number of 10 ~ 7 virus was injected into the myocardium by local injection. The rats in AMI group and sham operation group were given equal volume of saline daily for 1 week for 2 consecutive weeks. Cardiac function was recorded. The apoptotic index (AI) was detected by TUNEL, the expression of miR-221 was detected by RT-PCR, and the protein expressions of Bax, Bcl-2, PI3K and p-AKT were detected by Western blot. Results Compared with the sham group, the miR-221 expression in the AMI group (1.16 ± 0.12) and the control group (1.18 ± 0.12) was significantly increased (all P <0.01 (0.30 ± 0.03) in the inhibition group were lower than those in the AMI group and the control group (all P <0.01). Compared with the sham operation group, the Bax (0.13 ± 0.01) , Bcl-2 (0.53 ± 0.05), PI3K (0.45 ± 0.04) and p-AKT (0.87 ± 0.09) in the AMI group and the control group were significantly increased (all P <0.01 Bcl-2, PI3K and p-AKT were significantly decreased (all P <0.01). The expression of Bax in both groups were lower than those in AMI group and control group (P <0.01) PI3K and p-AKT protein levels were up-regulated (P <0.01). Conclusion miR-221 is overexpressed in myocardium of AMI rats. Down-regulating the expression of miR-221 can inhibit the expression of Bax and Bcl-2 in PI3K / AKT signaling pathway and inhibit the apoptosis of myocardial cells in AMI rats.