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（Exos^mimic，终质量浓度 50 μg·mL^-1）组、miRNA 阴性对照修饰外泌体（Exos^NC，终质量浓度 50 μg·mL^-1）组和 miR-199a修饰外泌体＋蛋白激酶 B（Akt）抑制剂（Exos^mimic＋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（ΔΨm）和线粒体动力学变化；Western blotting法检测各组缺氧诱导因子1α（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，Exos^mimic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、ΔΨm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，Exos^mimic组细胞存活率和ATP、SOD和ΔΨm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Exos^mimic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。

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, Exos^mimic group (final concentration of 50 μg·mL^-1), Exos^NC group (final concentration of 50 μg·mL^-1), and Exos^mimic+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 (ΔΨm) 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 Exos^mimic 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 ΔΨm 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 Exos^mimic group showed a significant increase in cell survival rate, ATP, SOD, and ΔΨm 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 Exos^mimic effect (P <0.05, 0.01). Conclusion miR-199a modified MSCs exosomes exert mitochondrial protective effects by AKT/HIF-1α/DRP1 axis.

R363

国家自然科学基金资助项目（82004329）；天津市医学重点学科（专科）建设项目