Src家族激酶（SFK）介导干细胞募集和缺血心脏修复的作用机制

Bone-morrow stem/progenitor cell (BM SPC) therapy is a promising new approach for treating ischemic heart disease, but the efficacy has been modest. Major barriers to successful cell therapy include poor recruitment of cells from the BM to the site of injury and the low proportion of recruited cells that are retained and survive in the ischemic region. A large amount of evidence now suggests that the recruitment and retention of BM SPCs in the ischemic tissue are regulated by interactions between stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4). We have recently discovered that Src family kinase (SFKs) is a crucial downstream mediator of SDF-1/CXCR4 signaling, and that inhibition of SFK abrogates SDF-1/CXCR4-induced BM EPC migration and recruitment to the ischemic myocardium (Cheng M et al, J Mol Cell Cardiol 2015; 81: 49-53). However, it remains unknown whether SFK-mediated BM SPC recruitment augments vasculogenesis in the ischemic myocardium and whether the SDF-1/CXCR4–SFK pathway can be harnessed to enhance SPC-mediated cardiac repair and heart function. Our central hypothesis is that in SPCs, SDF-1/CXCR4 signaling induces SFK phosphorylation (activation), which in turn mediates SPC recruitment to the ischemic tissue, and that techniques that up-regulate SFK phosphorylation can sensitize SDF-1/CXCR4 signaling, enhancing SPC recruitment and cardiovascular repair. We will test our hypotheses by a series of experiments grouped under three specific aims. Aim 1 will determine the role of SFK in SDF-1/CXCR4–mediated BM SPC recruitment and vasculogenesis by adoptive transfer of SFK-deficient and wild-type BM SPCs into SDF-1–transgenic mice after surgically-induced myocardial infarction (MI). Aim 2 will dissect the molecular and cellular mechanisms of SDF-1/CXCR4–mediated SFK activation by demonstrating the dependence of SPC migration on SFK phosphorylation, the involvement of beta-arrestin in mediating SFK phosphorylation, and by identifying novel phospho-SFK–interacting proteins. Aim 3 will determine whether SPC recruitment and cardiac repair after MI can be improved by increasing SFK activity in BM SPCs. Successful completion of the proposed research will likely lead to a better understanding of BM SPC recruitment in the ischemic tissue and to more effective approaches for SPC-mediated cardiac repair and cell therapy.

干细胞治疗缺血性心脏病潜力巨大，其在缺血部位的招募和驻留不足是影响疗效的关键。研究表明SDF-1/CXCR4是骨髓干细胞动员和招募的关键信号，是目前公认的干细胞修复调节的重要靶点，但针对该靶点进行细胞治疗的早期临床试验疗效欠佳。我们课题组最近报道，SFK是SDF-1/CXCR4信号通路的重要下游信号分子。本项目拟深入探讨：1）在SDF-1转基因鼠的心梗模型上转输野生型及SFK缺陷型的骨髓干细胞，明确SFK在SDF-1/CXCR4介导的干细胞向缺血心脏招募中的作用；2）鉴定SDF-1/CXCR4调节SFK磷酸化的新的信号分子，阐明其调节干细胞迁移的分子细胞机制；3）上调干细胞内SFK的活性，增加其对SDF-1/CXCR4信号的敏感性，从而促进其招募到缺血心脏组织。明确以SFK为新靶点的干细胞修复调节通路，克服目前细胞治疗的瓶颈，促进干细胞介导的缺血损伤修复和细胞治疗的疗效。

急性心肌梗死（AMI）后，心肌源性microRNAs（myocardial microRNAs，myo-miRs）在外周循环中显著升高，但其效应靶器官及功能学作用尚不清楚。循环中的外泌体是一类由细胞主动性分泌的具有细胞膜结构的小型运输体，能介导不同细胞或组织间mRNA、microRNA等遗传物质的交换。目前已知急性心梗能介导骨髓干细胞的动员。因此，本研究拟探讨急性心肌梗死后循环中升高的myo-miRs是否通过外泌体远程传输到骨髓，从而发挥对骨髓中干/祖细胞的动员作用。.研究结果发现：（1）急性心肌梗死患者外周循环中myo-miRs (miR-1a,133a,208a,499-5p)在急性心肌梗死患者中6h开始显著升高。升高的myo-miRs主要转运到骨髓。（2）急性心梗患者外周血中exosome与non-exosome中myo-miRs的含量在exsome中显著增多。（3）急性心梗分离的外泌体和非外泌体血清成份跟骨髓单核细胞共培养，发现有外泌体共培养的骨髓单核细胞中myo-miRs的表达增高，说明主要是由外泌体介导了循环中myo-miRs向骨髓单核细胞的转运。SDF-1/CXCR4信号通路在骨髓干细胞动员中起重要作用。在体外转染myo-miRs的特异性化学模拟物（microRNA mimics）到骨髓单核细胞中，发现这四种myo-miR均对CXCR4有明显抑制作用。（4）为了步验证急性心梗分泌的外泌体是否通过抑制CXCR4介导了骨髓干细胞的动员，在小鼠体内静脉注射急性心肌梗外泌体及正常对照外泌体，结果发现在急性心肌梗死外泌体处理的小鼠外周血中干/祖细胞显著增多，更进一步证明了外泌体转运miRNA到骨髓，介导了干细胞的动员并通过CXCR4调节。本研究首次提出心肌分泌的miRNA参与调节了骨髓干细胞的动员，具有重要的临床意义，为干细胞介导的缺血心肌修复提供了新的治疗靶点。