远端缺血预处理诱导的外泌体递送miR-24减轻再灌注心肌损伤及其机制研究

Remote ischemic preconditioning (RIPC) is well appreciated as a protector of myocardium against ischemia/reperfusion injury (IRI), but the underling mechanism remain elusive. Our previous studies evidenced that pro-apoptosis protein Bim is involved in myocardial injury through multiple ways, while miR-24 has myocardial protective effect via down-regulating Bim expression. Furthermore, our preliminary data indicated that exosomes in plasma carried more miR-24 after the animal treated with RIPC. Based on these, we put forward the hypothesis that exosomes induced by RIPC may transport miR-24 to attenuate myocardial IRI. We propose to apply series experiments in in vitro and in vivo, such as cell culture, confocal microscopy, IRI and RIPC animal models to verify that exosomes protect heart from IRI; and utilize luciferase assay, over-expression or knock-down of miR-24 in cardiomyocytes, RT-PCR and Western blot to verify that exosomes achieve their cardioprotective function through transferring miR-24 to reduce the expression of Bim in cardiomyocytes. In short, this study will make a useful exploration to verify the theory that exosomes induced by RIPC are important cardioprotective factors.

远端缺血预处理(RIPC)对缺血再灌注损伤（IRI）心肌有良好的保护作用，但具体机制未明。我们前期证实促凋亡蛋白Bim通过多种途径导致心肌损伤，而miR-24可以下调Bim从而具有心肌保护作用。预实验结果显示RIPC动物血浆中外泌体携带的miR-24明显升高。据此，我们提出假说：RIPC诱导的外泌体携带转运miR-24可以减轻再灌注心肌损伤。我们拟建立心肌细胞缺氧复氧模型及大鼠心脏离体灌注模型，通过细胞存活率检测、心肌梗死面积测定、生化检测等体内外实验证实RIPC诱导的外泌体对心肌的保护作用；通过激光共聚焦显微镜检查、荧光素酶实验、miR-24过表达及抑制实验、凋亡及自噬检测、RT-PCR及蛋白印迹等分子生物学实验证实外泌体的心肌保护作用是通过递送miR-24下调靶蛋白Bim表达而实现的。最终证实RIPC诱导的外泌体是重要的心肌保护体液因子，为深入理解RIPC的心肌保护机制做有益的探索。

众所周知，远端缺血预处理（RIPC）可保护心肌免受缺血/再灌注损伤（IRI）。外泌体是细胞外囊泡的一种，目前已成为细胞间通讯的关键介质。各种研究已证实循环外泌体能够介导RIPC。但是，RIPC诱导的外泌体介导的心脏保护的基本机制仍然不清楚。在我们的研究中，我们发现miR-24在RIPC所致大鼠血浆中的外泌体中的表达水平高于对照大鼠中血浆中的外泌体。研究进一步表明 H9c2细胞可吸收大鼠血浆外泌体。此外，我们还发现RIPC诱导的外泌体能够通过传递miR-24从而下调H9c2细胞的促凋亡蛋白Bim表达，在减轻氧化应激介导的损伤和减少细胞凋亡中发挥了重要作用。在在体实验中，RIPC诱导的外泌体中减少了心肌细胞的凋亡，减轻了梗塞面积并改善了心脏功能。此外，miR-24抑制剂在体内外均抵消了miR-24的凋亡减少作用。因此，我们表明，RIPC诱导的外泌体可以通过旁分泌方式转移miR-24来减少细胞凋亡，并且外泌体中的miR-24在介导RIPC的保护作用中起着核心作用。