miR-22修饰的骨髓间充质干细胞外泌体抑制急性心肌梗死大鼠心肌细胞凋亡

目的探讨miR-22修饰的骨髓间充质干细胞(BMSCs)外泌体对急性心肌梗死(AMI)大鼠心肌细胞凋亡的作用及其机制。方法扩增培养从SD大鼠骨髓中分离的BMSCs,将携带miR-22重组慢病毒载体及其阴性对照(miR-NC)载体分别转染至BMSCs,并提取转染后BMSCs的外泌体。将60只SD大鼠随机分为Sham组、AMI组、Exo BMSCs组、Exo BMSCs/miR-NC组和Exo BMSCs/miR-22组,每组12只。采用冠状动脉左前降支结扎术构建AMI大鼠模型,于心肌梗死区原位注射外泌体进行干预。干预72 h后,采用超声心动图监测大鼠心脏功能;TTC染色观察大鼠心肌梗死情况;TUNEL染色观察心肌细胞凋亡水平;qRT-PCR检测心肌组织中miR-22及p53 mRNA表达水平;Western blot检测心肌组织中B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、天冬氨酸特异性半胱氨酸蛋白酶剪切体(Cleaved-caspase-3)及p53蛋白表达水平。结果与Sham组比较,AMI组大鼠心脏功能明显降低(P <0.05),心肌梗死面积(%:1.16±0.84 vs.38.68±5.31)和心肌细胞凋亡率(%:9.04±1.95 vs.66.70±5.92)明显增加(P <0.05),心肌组织中miR-22和Bcl-2蛋白表达水平明显降低(P <0.05),而p53、Bax和Cleavedcaspase-3等蛋白表达水平明显升高(P <0.05);与AMI组比较,各外泌体干预组大鼠心功能明显改善(P <0.05),心肌梗死面积(%:38.68±5.31 vs.23.54±3.94,22.33±4.12,13.79±2.25)和心肌细胞凋亡率(66.70±5.92 vs.43.73±3.39,42.67±2.87,30.73±2.97)明显降低(P <0.05),心肌组织中miR-22以及Bcl-2蛋白表达水平明显升高(P <0.05),而p53、Bax和Cleaved-caspase-3等蛋白表达水平明显降低(P <0.05),其中Exo BMSCs/miR-22组大鼠各指标改善情况均优于Exo BMSCs组和Exo BMSCs/miR-NC组。结论 miR-22修饰的BMSCs外泌体可有效抑制p53介导的心肌细胞凋亡,从而改善AMI大鼠的心功能。

Effect of the exosomes of bone marrow mesenchymal stem cells modified by miR-22 on cardiomyocyte apoptosis in the rats with acute myocardial infarction

Objective To investigate the effect of exosomes of bone marrow mesenchymal stem cells( BMSCs) modified by miR-22 on cardiomyocyte apoptosis in acute myocardial infarction(AMI)rats and its mechanism. Methods BMSCs isolated from the bone marrow of SD rats were amplified and cultured. BMSCs were infected with miR-22 recombinant lentivirus vector and miR-NC vector respectively,and exosomes of the infected BMSCs were extracted. 60 SD rats were randomly divided into Sham group,AMI group,Exo BMSCs group,Exo BMSCs/miR-NC group and Exo BMSCs/miR-22 group,12 rats in each group. AMI rat models were established by the ligation of anterior descending branch of the left coronary artery. Exosomes were injected into the infarcted area in situ for intervention.After 72 hours of intervention,echocardiography was used to monitor the cardiac function. TTC staining was used to observe the myocardial infarction. TUNEL staining was used to observe the apoptosis level of myocardial cells. qRT-PCR was used to detect the expression level of miR-22 and p53 mRNA in myocardial tissue. Western blot was used to detect the expression level of B-cell lymphoma-2( Bcl-2),Bcl-2 associated X protein( Bax),cleaved cysteinyl aspartate specific protease-3( Cleaved-caspase-3) and p53 protein in myocardial tissue. Results Compared with the Sham group,myocardial function was significantly decreased in the AMI group( P < 0. 05),infarction area[( 1. 16 ±0. 84) % vs.( 38. 68 ± 5. 31) % ] and cardiomyocyte apoptosis rate[( 9. 04 ± 1. 95) % vs.( 66. 70 ±5. 92) %] were significantly increased( P < 0. 05),and the expression levels of miR-22 and Bcl-2 in myocardial tissue were significantly decreased( P < 0. 05),while the protein expression levels of p53,Bax and Cleaved-caspase-3 were significantly increased( P < 0. 05). Compared with the AMI group,the cardiac function of rats in each exosomes intervention group was significantly improved( P < 0. 05),myocardial infarction area[AMI:( 38. 68 ± 5. 31) %,Exo BMSCs:( 23. 54 ± 3. 94) %,Exo BMSCs/miR-NC:( 22. 33 ± 4. 12) %,Exo BMSCs/miR-22:( 13. 79 ± 2. 25) %]and apoptosis rate of myocardial cells[AMI:( 66. 70 ± 5. 92) %,Exo BMSCs:( 43. 73 ± 3. 39) %,Exo BMSCs/miR-NC:( 42. 67 ± 2. 87) %,Exo BMSCs/miR-22:( 30. 73 ± 2. 97) %] were significantly reduced( P <0. 05),and the expression levels of miR-22 and Bcl-2 in myocardial tissue were significantly increased( P < 0. 05),while the protein expression levels of p53,Bax and Cleaved-caspase-3 were significantly decreased( P < 0. 05),and the improvement of each index in the Exo BMSCs/miR-22 group was more apparent than that in the Exo BMSCs group and Exo BMSCs/miR-NC group.Conclusion The exosomes of BMSCs modified by MiR-22 can effectively inhibit the apoptosis of cardiomyocytes mediated by p53,thus improve the cardiac function of AMI rats.