Notch1与RISK/SAFE/HIF-1α信号通路整合在I-postC保护中的作用及其机制

The ischemic postconditioning (I-postC) provides protection.from myocardial injury by an unknown mechanism. Based on our preliminary absorvation.and previous report, we hypothesize that Notch1 plays an important role in I-postC.by cross talking with RISK/SAFE/HIF-1α through NICD/Hes1. By technologies including.adenovirus vector construction, we plan to establish transgenic cell and rat based.on Notch1→NICD→Hes→PTEN or Dnase I→mitochondria. After confirming the effect.of I-postC in these experimental models, we will study the effect of Notch1 and Hes1.expression level on RISK/SAFE/HIF-1α downstream molecules p-Akt, p-Stat3, o-PTEN.and DNase I. We will examine the interactions between N1ICD, Hes1, and Akt, Stat3,.HIF-1α, and the subcellular location. Based on functional parameters including mPTP.opening, we will explore the crosstalk effect of aboving cell signaling pathways.on mitochondrial function, and test it in animal model. This study will clarify the.mechanism and significance of Notch1 signaling in I-postC, and identify a novel.target to develop new myocardial protection drugs.

在总结前人与自身工作基础上，我们假设"Notch1通过NICD/Hes1等因子，作为共同环节参与RISK/SAFE/HIF-1α信号通路整合，在I-postC中发挥重要作用"，围绕"Notch1→NICD→Hes1→PTEN或DnaseⅠ→线粒体功能改变"主线，采用腺病毒载体构建等技术，制备相应转基因细胞与整体大鼠；确认已产生I-postC保护作用后，研究Notch1与Hes1不同表达水平对RISK/SAFE/HIF-1α等信号通路下游p-Akt、p-Stat3、o-PTEN及DNaseⅠ影响；探讨N1ICD、Hes1与Akt、Stat3、HIF-1α间交互作用以及在细胞内的亚定位；通过mPTP开放等功能指标，追溯上述信号通路间整合对细胞线粒体功能影响；并在整体动物比较论证。可望阐明其在I-postC心肌保护意义及相关分子机制，且可作为新的靶点，为研制新型心肌保护药物奠定的理论与实验基础。

我们依据“Notch1 通过NICD/Hes1 等因子，作为共同环节参与RISK/SAFE/HIF-1α信号通路整合，在I-postC中发挥重要作用”假设，围绕“Notch1→NICD→Hes1→PTEN 或DnaseⅠ→线粒体功能改变”主线，在确认Notch1信号通路在心肌细胞H/R、IPC、IPost中激活基础上，构建Notch1及Hes1腺病毒表达与干扰载体，制备Notch1及Hes1转基因细胞与整体大鼠，建立缺血再灌注、缺血预适应、缺血后适应细胞与动物模型，依次检测心肌细胞存活率、心肌细胞凋亡、活性氧、心肌酶谱、心肌梗死面积及左心功能改变，观察Notch1信号通路的心肌保护效应，Western blot检测PTEN、p-Akt/Akt、p-GSK-3β/GSK-3β、p-Stat3/Stat3、Bax、Bcl-xl、HIF-1α、DnaseI蛋白表达，从中探讨Notch1信号通路与RISK/SAFE/HIF-1α信号通路相互整合作用；分离线粒体，Western blot检测线粒体/细胞质CytC表达差异，流式细胞仪检测线粒体膜电位，从而追溯上述信号通路间整合对细胞线粒体功能影响。我们实验数据表明Notch1作为内源性心肌保护因子，通过与RISK/SAFE/HIF-1α信号通路相互整合，减少心肌细胞内活性氧产生，稳定线粒体膜电位，抑制线粒体通透性转换孔开放，减缓心肌酶谱释放，从而增强心肌细胞活力，减轻心肌细胞凋亡，缩小心肌梗死面积，改善左心功能，明显减轻心肌缺血再灌注损伤。该课题已阐明Notch1内源性心肌保护作用意义及相关分子机制，并可作为新的靶点，为研制新型心肌保护药物奠定的理论与实验基础。