摘要
目的 探讨miR-199a修饰的间充质干细胞(MSCs)来源的外泌体修复缺糖缺氧/复糖复氧模型心肌细胞H9c2线粒体的作用机制.方法 体外培养MSCs,转染miR-199a mimics或miR-NC,48～72 h后收集外泌体,实时荧光定量PCR(qRT-PCR)法检测外泌体的miR-199a水平.将H9c2细胞分为对照组、模型组、miR-199a修饰外泌体(Exosmimie,终质量浓度50μg·mL-1)组、miRNA阴性对照修饰外泌体(ExosNC,终质量浓度50μg·mL-1)组和miR-199a修饰外泌体+蛋白激酶B(Akt)抑制剂(Exosmimic+MK2206 10 μg·mL-1)组,除对照组外,制备缺糖缺氧/复糖复氧模型.应用CCK-8法检测各组细胞存活率,酶标仪检测各组细胞三磷酸腺苷(ATP)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平以及上清液8-羟基脱氧尿苷(8-OHdG)、乳酸脱氢酶(LDH)水平;共聚焦显微镜检测各组线粒体膜电位(△Ψm)和线粒体动力学变化;Western blotting法检测各组缺氧诱导因子lα(HIF-1α)和线粒体动力相关蛋白1(DRP1)蛋白表达变化.结果 与对照组比较,Exosmimic中miR-199a表达水平明显升高(P＜0.05),提取的外泌体直径平均为109.3 nm,浓度为1× 106颗粒·mL-1.与对照组比较,模型组细胞存活率和ATP、SOD、△Ψm水平显著下降,LDH、MDA、8-OHdG水平和HIF-1α和DRP1蛋白表达水平显著升高(P＜0.01、0.001),线粒体分裂水平增加;与模型组比较,Exosmimic组细胞存活率和ATP、SOD和AΨm水平显著升高,LDH、MDA、8-OHdG水平和HIF-1α及DRP1蛋白表达水平显著下降(P＜0.01、0.001),线粒体分裂水平减少;MK2206能明显逆转Exosmimic效果(P＜0.05、0.01).结论 miR-199a修饰的MSCs外泌体通过Akt/HIF-1α/DRP1轴促进缺糖缺氧/复糖复氧心肌细胞线粒体修复.
Abstract
Objective To investigate the mechanism of miR-199a-modified mesenchymal stem cell(MSC)-derived exosomes in repairing mitochondria of H9c2 cardiomyocytes in a model of hypoglycemia/hypoxia/reoxygenation with hypoglycemia.Method Transfect miR-199a mimics or miR-NC into MSCs and collect exosomes 48-72 h later.Fluorescence quantitative PCR(qRT-PCR)method was used to detect miR-199a levels in different exosomes.H9c2 cells were divided into control group,model group,Exosmimic group(final concentration of 50 μg·mL-1),ExosNC group(final concentration of 50 µg·mL-1),and Exosmimic+MK2206(10 μg·mL-1)group.Except for the control group,the model of hypoglycemia/hypoxia/reoxygenation with hypoglycemia was established.The survival rate of cells in each group was detected by CCK8 assay,and the levels of ATP,8-hydroxy-2-deoxyguanosine(8-OHdG),lactate dehydrogenase(LDH),superoxide dismutase(SOD),malondialdehyde(MDA)in each group were detected by enzyme-linked immunosorbent assay(ELISA).The changes of mitochondrial membrane potential(AΨm)and dynamics were detected by confocal microscopy.The expression changes of hypoxia-inducible factor 1α(HIF-1α)and mitochondrial dynamics-related protein 1(DRP1)were detected by Western blotting.Result Compared with control group,the expression level of miR-199a in Exosmimicc was significantly increased(P＜0.05),and the average diameter of extracted exosomes was 109.3 nm,with a concentration of 1×106 particles·mL-1.Compared with control group,the model group showed a significant decrease in cell survival rate and ATP,SOD,and AΨm levels,while LDH,MDA,8-OHdG levels,HIF-1α and DRP1 protein expression levels were significantly increased(P＜0.01,0.001),and mitochondrial division levels were increased.Compared with model group,the Exosmimic group showed a significant increase in cell survival rate,ATP,SOD,and AΨm levels,while LDH,MDA,8-OHdG levels,HIF-1α,and DRP1 protein expression levels were significantly decreased(P＜0.01,0.001),and mitochondrial division levels were reduced.MK2206 can significantly reverse the Exosmimic effect(P＜0.05,0.01).Conclusion miR-199a modified MSCs exosomes exert mitochondrial protective effects by AKT/HIF-1α/DRP1 axis.
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