目的 探討miR-199a修飾的間充質(zhì)干細(xì)胞（MSCs）來源的外泌體修復(fù)缺糖缺氧/復(fù)糖復(fù)氧模型心肌細(xì)胞H9c2線粒體的作用機制。方法 體外培養(yǎng) MSCs，轉(zhuǎn)染 miR-199a mimics 或 miR-NC，48～72 h 后收集外泌體，實時熒光定量PCR（qRT-PCR）法檢測外泌體的 miR-199a水平。將 H9c2細(xì)胞分為對照組、模型組、miR-199a修飾外泌體（Exos^mimic，終質(zhì)量濃度 50 μg·mL^-1）組、miRNA 陰性對照修飾外泌體（Exos^NC，終質(zhì)量濃度 50 μg·mL^-1）組和 miR-199a修飾外泌體＋蛋白激酶 B（Akt）抑制劑（Exos^mimic＋MK2206 10 μg·mL^-1）組，除對照組外，制備缺糖缺氧/復(fù)糖復(fù)氧模型。應(yīng)用 CCK-8 法檢測各組細(xì)胞存活率，酶標(biāo)儀檢測各組細(xì)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-羥基脫氧尿苷（8-OHdG）、乳酸脫氫酶（LDH）水平；共聚焦顯微鏡檢測各組線粒體膜電位（ΔΨm）和線粒體動力學(xué)變化；Western blotting法檢測各組缺氧誘導(dǎo)因子1α（HIF-1α）和線粒體動力相關(guān)蛋白1（DRP1）蛋白表達變化。結(jié)果 與對照組比較，Exos^mimic中 miR-199a 表達水平明顯升高（P＜0.05），提取的外泌體直徑平均為 109.3 nm，濃度為 1×10⁶顆?！L^-1。與對照組比較，模型組細(xì)胞存活率和ATP、SOD、ΔΨm水平顯著下降，LDH、MDA、8-OHdG水平和HIF-1α和DRP1蛋白表達水平顯著升高（P＜0.01、0.001），線粒體分裂水平增加；與模型組比較，Exos^mimic組細(xì)胞存活率和ATP、SOD和ΔΨm水平顯著升高，LDH、MDA、8-OHdG水平和HIF-1α及DRP1蛋白表達水平顯著下降（P＜0.01、0.001），線粒體分裂水平減少；MK2206能明顯逆轉(zhuǎn)Exos^mimic效果（P＜0.05、0.01）。結(jié)論 miR-199a修飾的MSCs外泌體通過Akt/HIF-1α/DRP1軸促進缺糖缺氧/復(fù)糖復(fù)氧心肌細(xì)胞線粒體修復(fù)。

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.

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國家自然科學(xué)基金資助項目（82004329）；天津市醫(yī)學(xué)重點學(xué)科（?？疲┙ㄔO(shè)項目