CaMEK基因靶向干预防治糖尿病心脏缺血再灌注损伤的机制研究

Diabetes is a risk equivalents of coronary heart disease. According to db/db mice study we found that pathological changes of diabetes increase the sensitivity of myocardial ischemia-reperfusion injury, and myocardial ERK 1/2 signaling pathways were significantly lower than the control group, while the mechanism is still unclear. CaMEK gene can specifically activate the ERK1/2 signaling pathway and protect against myocardial ischemic injury. Accordingly, the present study is going to apply myocardial ischemia-reperfusion injury model to investigate the mechanism of ERK1/2 pathway in diabetic myocardial ischemia-reperfusion injury. While, a fast double-stranded recombinant gene expression of CaMEK AAV9 vector will be built via in vivo and in vitro transfection experiments at the cellular, tissue and whole animal level, to identify the potential regulatory mechanisms of CaMEK on key target gene of ERK1/2 pathway. Based on exogenous CaMEK transduced into db/db diabetic mice, we try to illustrate treatment effects and molecular mechanisms of targeting myocardial activation of ERK1/2 signaling pathway in diabetic ischemia-reperfusion myocardial injury, and determine therapeutic effect of exogenous CaMEK as a target in diabetic myocardial ischemia. This study aims to provide new ideas and basis for alleviating diabetic ischemia-reperfusion injury and improve the prognosis of diabetic patients with coronary heart disease.

糖尿病是冠心病的等危症，我们db/db小鼠研究发现：糖尿病病理变化加重心肌缺血再灌注损伤敏感性，且心肌ERK1/2信号通路活性较对照组显著降低，但机制不清楚。CaMEK 基因能特异性激活ERK1/2信号通路，抵抗心肌缺血损伤。据此，本课题拟采用心肌缺血再灌注损伤模型，探讨ERK1/2通路在糖尿病心肌缺血再灌注损伤中的作用机制；同时，构建快速表达CaMEK基因的双链重组AAV9载体，通过体内外转染实验，在细胞、组织及动物整体水平上，明确CaMEK对ERK1/2通路关键靶基因的调控机制；基于db/db糖尿病小鼠体内转导外源性CaMEK干预实验，阐明靶向激活心肌ERK1/2信号通路防治糖尿病心肌缺血再灌注损伤的效果及分子机制，确定外源CaMEK作为糖尿病心肌缺血损伤治疗靶标的效用，为减轻合并糖尿病的冠心病患者缺血再灌注损伤、改善预后提供新的思路和依据。

本项目针对糖尿病患者心肌对缺血再灌注损伤尤为敏感，治疗效果不理想的临床现象，首次阐明ERK1/2磷酸化水平没有被缺血后适应显著激活，是后适应糖尿病心肌保护作用消失的关键节点，ERK1/2信号通路的活性缺失导致的心肌细胞凋亡显著增加了糖尿病心肌缺血再灌注损伤性。以MEK为靶基因，通过基因重组技术构建双链AAV9-CaMEK载体，揭示CaMEK基因靶向转导减轻糖尿病心肌缺血再灌注损伤的分子机制，在器官水平上评价 dsAAV9 载体 表达的组织特异性。研究发现该病毒可高效靶向的在糖尿病小鼠心脏表达并发挥调控作用。建立糖尿病小鼠缺血再灌注模型及心肌细胞缺氧复氧模型，发现转染双链重组9型腺相关病毒载体可以靶向激活心肌ERK1/2信号通路。在糖尿病小鼠心肌缺血再灌注的情况下，AAV9-CaMEK载体转导可以减少糖尿病小鼠心肌梗死面积，改善心功能，减少细胞凋亡,研究发现该机制可能通过调控线粒体动力学相关蛋白完成。细胞学机制研究也发现AAV9介导的CaMEK基因转染心肌细胞增强了磷酸化ERK1/2蛋白的表达，并因此上调p70S6K、GSK3β的表达进一步调控心肌细胞凋亡。并为今后患糖尿病的冠心病患者心肌缺血再灌注损伤提供思路和依据，具有潜在的临床应用前景和研究价值。