miR-454/SDF-1/CXCR4通路在重复远程缺血后适应调节间充质干细胞经心外膜移植后脏器再分布的作用研究

Coronary artery disease is the main disease that endanger the health of the citzens in our country. There had been some clinical trials which testified that mesenchymal stem cells transplantation was an effective method to treat ischemic heart disease. However, its effcacy was restricted because the part of mesenchymal stem cells via intramyocardial transplantation entered into bloodflow and migrated to peripheral solid organs.Remote ischemic postconditioning (RIPoC) is an endogenous protective strategy with the characteristic of non-invasive, effectivity and repeatability. It had previously been demonstrated in our research that RIPoC activated stromal cell derived factor-1 (SDF-1) gene expression in chronic ischemic myocardium. SDF-1 is characteristic of recruiting stem cells to ischemic myocardium to repair the injuried heart. Based on previous research, micro-RNA 454 control the gene expression of SDF-1.This study is to investigate whether repeated RIPoC (rRIPoC) decreased the micro-RNA 454 level in ischemic mycardium, and thereof negatively regulated its targeted gene SDF-1 expression level, therefore, rRIPoC decreased the MSCs retention percentage in extracardiac organs which had migrated from heart, enhanced the intramyocardially transplanted MSCs retention in ischemia-reperfused myocardium, and improved cardiac function from the facets of the molecular, cell and organ on the rat heart ischemia reperfusion model. The aim of this study is to enhance therapeutic efficacy of MSCs transplantation from a new perspective of changing micro-environment by modifing recipent, so as to introduce ischemic conditioning into clinical trials on cell transplantation. Meanwhile, it is also to elucidate the new signalling pathway on the inner mechanism of ischemic conditioning.

冠心病是危害国民健康的主要疾病。已有临床试验显示间充质干细胞(MSCs)移植治疗缺血性心肌病是一种有效的方法。然而经心外膜注射移植时部分细胞进入血液循环迁移至外周实体脏器限制了其疗效。远程缺血后适应(RIPoC)是一种无创、有效、可重复的内源性心肌缺血保护策略。前期工作显示RIPoC激活慢性缺血心肌的基质细胞衍化因子-1(SDF-1)表达。SDF-1具有募集干细胞到缺血心肌的重要作用。有研究表明microR-454可调控SDF-1的表达。本研究在大鼠心肌缺血再灌注模型中从分子、细胞、器官水平研究重复远程缺血后适应(rRIPoC)是否通过microR-454/SDF-1/CXCR4轴募集迁移分布在外周器官的MSCs到缺血心肌，改善心脏功能。该研究从修饰移植受体微环境增加细胞迁移的角度改善MSCs的移植效率，以期实现缺血性适应用于细胞移植临床试验，同时阐述缺血性适应内在作用机制新的信号通路。

缺血性心脏病是当今人群主要易患疾病之一。间充质干细胞移植是一种治疗缺血性心脏病新模式。远程缺血后适应(RIPoC)是一种无创、有效、可重复的内源性心肌缺血保护策略。我们在之前的研究发现研究重复远程缺血后适应（rRIPoC）可以增加间充质干细胞（MSC）移植治疗缺血性心脏病的疗效。本项目我们探索rRIPoC的实验作用效应机制。首先，提取大鼠血浆外泌体，通过micro-RNA芯片检测发现miR-122-5p在重复远程缺血后适应、远程缺血后适应、心梗组、对照组之间差异明显。并通过RT-PCR实验验证血浆中rRIPoC组的miRNA-122-5p表达升高。随后，通过共培养实验检测发现MSC可以摄取外泌体。经过缺血缺氧处理的MSC活性下降，加入rRIPoC处理提取的外泌体后，细胞活性改善，且效果与加入miRNA-122-5p agomir的结果类似，再以miRNA-122-5p antoagomir处理后，细胞活性又被抑制。从时间效应上来看，以上情况在rRIPoC处理后48和72h有显著性差异。通过检测MSC的干性（Nanog，Oct-4）和衰老（P16，P53）情况，发现加入rRIPoC处理的外泌体及miRNA-122-5p agomir均可改善MSC的干性和衰老标记。另外，经过缺血缺氧处理的MSC细胞的γ-H2AX表达显著上升，加入rRIPoC处理的外泌体及miRNA-122-5p agomir均可改善提示rRIPoC处理通过miRNA-122-5p改善MSC的DNA损伤。为了检验缺血性适应在心血管疾病中临床应用价值。我们通过临床试验发现远程缺血预适应可以增加体外循环下心房纤颤外科手术转复窦性心律的成功率。在模拟缺血缺氧适应的临床模式中，出生后高原居住缺氧性适应可以改善心脏瓣膜手术后的缺血再灌注损伤的炎症反应、心肌损伤指标，以及临床功能恢复指标。该项目的科学意义及研究价值在于从miRNA测序筛查以及验证出发，寻找缺血性适应的靶向调控，有助于确定缺血性适应的作用机制以及干细胞移植的干预目标。应用缺血性适应在心脏外科临床手术治疗风心病心房纤颤患者具有改善心律转复的作用，表明缺血性适应在心律失常治疗中的积极意义。发现出生后低氧环境具有抗心肌缺血再灌注损伤作用，对评估高原缺氧人群在心脏瓣膜手术治疗过程中病情具有重要临床指导价值。