Myocardin相关转录因子-A对缺氧缺血诱导的大鼠心肌细胞凋亡的影响

Protection effect of Myocardin-related transcription factor-A on myocardial survival via inhibiting apoptosis

目的研究Myocardin相关转录因子-A(MRTF-A)对缺氧缺血诱导的心肌细胞凋亡的影响及机制。方法 20只大鼠采用随机数字表分为假手术组和缺血再灌注组。假手术组大鼠实行开胸手术但不构建缺血再灌注模型,缺血再灌注组结扎冠状动脉30min再松开2h建立在体心肌缺血再灌注损伤模型。采用TUNEL法检测心肌细胞凋亡,免疫组化法检测MRTF-A表达;体外培养大鼠原代心肌细胞,转染MRTF-A及小分子mRNA表达质粒并构建缺氧缺血模型。通过流式细胞术检测细胞凋亡率,以Western blot法分析凋亡相关基因bcl-2蛋白表达;采用荧光素酶检测bcl-2转录活性。结果假手术组和缺血再灌注组的心肌凋亡细胞分别为(8.14±2.33)%和(82.35±7.93)%,缺血再灌注组高于假手术组(P<0.05)。假手术组有少量MRTF-A蛋白表达,缺血再灌注组胞浆中有大量MRTF-A蛋白表达。在体外培养的原代心肌细胞中,对照组、模型组、MRTF-A处理组与MRTF-A抑制组的凋亡率分别为(4.63+2 33)%、(79.97+9.09)%、(43.63+3.52)%和(72.16+5.37)%;bcl-2的蛋白表达分别为0.313+0.014、0.186+0.019、0.254+0 013与0.175+0.017。模型组凋亡率高于对照组(P<0.01),MRTF-A处理组的凋亡率低于模型组(P<0.01);MRTF-A抑制组的凋亡率高于MR:TF-A处理组(P<0.05);模型组bcl-2的蛋白低于对照组(P<0.01),MRTF-A处理组的bcl-2的蛋白高于模型组(P<0.01);MRTF-A抑制组的bcl-2的蛋白低于MRTF-A处理组(P)<0.05);MRTF-A对bcl-2的转录活性的调控与对照组相比明显升高(P<0.01),而MRTF-A对突变后的bcl-2的转录活性与对照组比较差异无统计学意义(P>0.05)。结论缺血再灌注损伤诱导了大鼠心肌细胞凋亡,上调了心肌组织中MRTF-A的蛋白表达;MRTF-A通过上调bcl-2的转录活性表达起到对缺氧缺血诱导的大鼠原代心肌细胞凋亡损伤的保护作用。

Objective To observe the effect of myocardial myocardin-related transcription factor-A （MRTF-A） on my- ooardium apoptosis and its mechanism. Methods 20 rats were randomly divided into sham and ischemia-reperfusion（myocar- dial ischemia30-min and reperfusion 2h） group （n=10 each）. TUNEL was used to detect cardiomyocyte apoptosis, the MRTF-A expression was detected by immunohistochemistry. One clay neonatal rat ventrical was used as cell culture and hypoxia-no glu- cose damaged myocardial cell before over-expression of wild-type MRTF-A or siRNA in myocardial cell. The apoptosis rate was measured by FCM. The bcI-2 expression was analyzed by western blot. The bcI-2 transcriptional activity was detected by lu- ciferase. Results The number of apoptotic cardiomyocytes in the sham group and ischemia-reperfusion group were （8.14_＋ 2.33）% and（82.35 ＋ 7.93）%, respectively （P〈0.05）. The protein expression of MRTF-A in the sham group were significantly lower than those in ischemia-reperfusion group. In cultured neonatal rat cardiomyocyte, the apoptotic of control group, model group, MRTF-A transfection group and siRNA transfection group were （4.63＋2.33）%, （79.97＋9.09）%, （43.63＋3.52）%, （72.t6＋5.37）%; The protein expression of bcl-2 were 0.313＋0.014,0.186＋0.019,0.254＋0.013,0.175＋0.017. Over-expression of wild-type MRTF-A upregulated the transcription activity of bcI-2 reporter gene, but the mutation bcI-2 reporter gene. Conclusion The expression of MRTF-A had changed that induced by isohemia-reperfusion. MRTF-A promoting myocardial survival against apoptosis induced by hypoxia-no glucose via up-regulation bcI-2 transcription activity.