骨髓间充质干细胞对氧化应激损伤心肌细胞凋亡、自噬的影响

Effect of bone marrow mesenchymal stem cell on apoptosis and autophagy of cardiomyocytes damaged by oxidative stress

目的探讨骨髓间充质干细胞(BMSC)对氧化应激损伤心肌细胞凋亡、自噬的影响。方法(1)以不同浓度(0μmol/L、50μmol/L、100μmol/L、300μmol/L、500μmol/L和700μmol/L)H 2O 2处理大鼠心肌细胞H9c26 h、9 h和12 h,采用CCK-8检测细胞活力,筛选H 2O 2的最佳作用浓度及作用时间以构建氧化应激损伤心肌细胞模型。(2)将大鼠心肌细胞H9c2分为对照组、H 2O 2组、BMSC+H 2O 2组。H 2O 2组和BMSC+H 2O 2组均采用500μmol/L H 2O 2诱导12 h建立氧化应激损伤心肌细胞模型,在此基础上,BMSC+H 2O 2组加入对数生长期大鼠BMSC进行共培养。对照组正常培养而不给予干预。采用流式细胞术检测各组细胞凋亡情况,分别采用实时荧光定量PCR、Western blot检测各组细胞自噬相关基因[Beclin-1、微管相关蛋白1轻链3β(LC3B)]mRNA和蛋白表达水平。结果与对照组相比,H 2O 2组细胞凋亡率升高(P<0.05);与H 2O 2组相比,BMSC+H 2O 2组细胞凋亡率降低(P<0.05)。与对照组、H 2O 2组比较,H 2O 2+BMSC组Beclin-1、LC3B mRNA和蛋白表达水平均升高(均P<0.05)。结论BMSC可以通过抑制心肌细胞凋亡及增强心肌细胞自噬水平,以减轻氧化应激对心肌细胞的损伤,从而发挥心肌保护作用。

Objective To investigate the effect of bone marrow mesenchymal stem cell(BMSC)on apoptosis and autophagy of cardiomyocytes damaged by oxidative stress.Methods(1)H9c2 cardiomyocytes of rats were treated with different concentrations of H 2O 2(0μmol/L,50μmol/L,100μmol/L,300μmol/L,500μmol/L,and 700μmol/L)for 6,9 and 12 hours.The CCK-8 was employed to detect cell viability for screening the optimal effect concentration and effect duration of H 2O 2 to establish cardiomyocyte model damaged by oxidative stress.(2)H9c2 cardiomyocytes of rats were assigned to control group,H 2O 2 group,or BMSC+H 2O 2 group.The H 2O 2 group and BMSC+H 2O 2 group used 500μmol/L H 2O 2 for 12-hour induction to establish the cardiomyocyte model damaged by oxidative stress,based on which in the BMSC+H 2O 2 group,BMSC of rats in logarithmic growth phase was added for co-culture.The control group received normal culture but without any intervention.Cell apoptosis of various groups was detected by employing flow cytometry,and the mRNA and protein expressions of autophagy-associated genes(Beclin-1,microtubule-associated protein 1 light chain 3β[LC3B])of cells in various groups were detected by using real-time fluorescent quantitative PCR and Western blot,respectively.Results Compared with the control group,the cell apoptosis rate in the H 2O 2 group was elevated(P<0.05).Compared with the H 2O 2 group,the cell apoptosis rate in the BMSC+H 2O 2 group was decreased(P<0.05).Compared with the control group and the H 2O 2 group,the BMSC+H 2O 2 group exhibited elevated mRNA and protein expressions of Beclin-1 and LC3B(all P<0.05).Conclusion BMSC through inhibiting apoptosis of cardiomyocytes and enhancing autophagy level of cardiomyocytes can relieve damage of oxidative stress on cardiomyocytes,so as to exert myocardial protection.