间充质干细胞来源的外泌体通过 microRNA-21-5p 调节心脏自噬并影响心肌缺血大鼠的心脏功能
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新疆医科大学第五附属医院心血管内科,乌鲁木齐 830011

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R-33

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Mesenchymal stem cell-derived exosomes regulate cardiomyocyte autophagy and cardiac function via microRNA-21-5p in rats with myocardial ischemia
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Department of Cardiovascular Medicine,Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, China

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    摘要:

    目的 探讨间充质干细胞(mesenchymal stem cells,MSCs)分泌的外泌体( exosome,Exos)是否通过 miR-21-5p 调节心脏自噬并影响心肌缺血大鼠的心脏功能。 方法 在体外,观察 MSCs-Exos 对 H2O2 刺激 H9C2s 的 影响;通过 CCK-8 测定法检测细胞活力;流式细胞术检测细胞凋亡;荧光显微镜检测细胞中活性氧( reactive oxygen species,ROS)的产生;免疫印迹分析自噬相关蛋白和荧光 GFP-LC3 测定自噬体形成。在大鼠 MI/ RI 模型中,通过 TUNEL 测定、免疫组织化学染色及超声心动图分别检查了 MSCs-Exos 对细胞凋亡、心肌 LC3B 表达和心脏功能影响。 结果 在体外实验中,MSCs-Exos 显著提高了 H2O2 刺激的 H9C2 细胞活力(P<0. 05),降低了 ROS 的产生和细 胞凋亡率(P<0. 05)。而与 H2O2 +MSCs-Exos 组相比,H2O2 +MSC-ExossimiR-21-5p组细胞活力显著降低(P<0. 01), ROS 的产生和细胞凋亡率显著增加(P<0. 01)。 Western blot 检测显示:与 H2O2 组相比,H2O2 +MSCs-Exos 组 LC3B- Ⅱ/ LC3B-Ⅰ和 LC3B-Ⅱ的表达显著增强(P<0. 01),p62 的表达显著降低(P<0. 01);与 H2O2 +MSCs-Exos 组相比, H2O2+MSC-ExossimiR-21-5p组的 LC3B-Ⅱ/ LC3B-Ⅰ和 LC3B-Ⅱ的表达显著降低(P<0. 05),p62 的表达显著增强(P< 0. 05)。自噬通量结果:与 H2O2 组比较,H2O2 +MSCs-Exos 组细胞中存在的 GFP-LC3 点数增加;而与 H2O2 +MSCs- Exos 组相比,H2O2+MSC-ExossimiR-21-5p组细胞中存在的 GFP-LC3 点数显著降低(P<0. 05)。 在体内,RT-qPCR 分析 结果显示:在 MSCs-Exos 中和 MI/ RI 后心肌组织中 miR-21-5p 的表达均呈正相关性。 与其他各组相比,MI/ RI + MSCs-Exos 组的 LC3B 表达显著增强(P<0. 01);心肌细胞凋亡显著减少(P<0. 01);分数缩短率(FS%)和左心室射 血分数(LVEF)均显著提高(P<0. 05)。 结论 MSCs-Exos 可通过调节心肌自噬改善 MI/ RI 大鼠的心脏功能,其作 用机制可能与 miR-21-5p 转移有关。

    Abstract:

    Objective To investigate whether exosomes (Exos) secreted by mesenchymal stem cells (MSCs) regulate cardiac autophagy and cardiac function in rats with myocardial ischemia through miR-21-5p. Methods In vitro,the effect of MSC exos on H2O2 stimulated H9C2 cells was observed. Cell viability was detected by the CCK- 8 assay, apoptosis was detected by flow cytometry, reactive oxygen species ( ROS ) production was analyzed by fluorescence microscopy, and autophagy-related proteins and autophagy formation were detected by immunoblotting and fluorescent GFP- LC3. In rat model of myocardial ischemia-reperfusion injury ( MI/ IR), the effects of MSC exos on cell apoptosis, myocardial LC3b expression, and cardiac function were examined by TUNEL assay, immunohistochemistry, and echocardiography. Results In vitro, MSC-Exos significantly increased the activity of H9C2s stimulated by H2O2(P<0. 05) and decreased the production of ROS and the rate of apoptosis (P<0. 05). Compared with the H2O2+MSCs-Exos group, the cell viability in the H2O2+MSC-ExossimiR-21-5p group was significantly decreased (P<0. 01), whereas ROS production and apoptosis were significantly increased (P<0. 01). Western blot analysis showed that compared with the H2O2 group, the expression of LC3B-Ⅱ/ LC3B-I and LC3B-II in the H2O2+MSC-ExossimiR-21-5p group was significantly lower than that in the H2O2+MSCs-Exos group (P<0. 05), while the expression of p62 was significantly increased (P<0. 05). Autophagy flux result : Compared with the H2O2 group, GFP-LC3 was increased in the H2O2+ MSCs-Exos group. Furthermore, Compared with the H2O2+ MSCs-Exos group, the number of GFP-LC3 points in the cells of the H2O2+ MSC-ExossimiR-21-5p group was significantly reduced (P<0. 05). in vivo, RT-qPCR analysis showed that there was a positive correlation between the expression of miR-21-5p in MSCs-Exos and MI/ RI. Compared with other groups, the expression of LC3B in the MI/ RI+ MSCs-Exos group was significantly increased (P<0. 01), myocardial apoptosis was significantly reduced (P<0. 01), and the fraction shortening rate (%FS) and left ventricular ejection fraction (LVEF) were significantly increased (P<0. 05). Conclusions MSCs-Exos can improve cardiac function in MI/ RI rats by regulating myocardial autophagy, and its mechanism may be related to miR-21-5p transfer.

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孙理华,王 娟,张 岳,张 颖,幸世峰.间充质干细胞来源的外泌体通过 microRNA-21-5p 调节心脏自噬并影响心肌缺血大鼠的心脏功能[J].中国比较医学杂志,2020,30(5):88~96.

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  • 收稿日期:2019-11-11
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  • 在线发布日期: 2020-06-19
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