内皮细胞A2A受体靶向介导游离皮瓣缺血后处理内源性抗凋亡机制研究

Ischemic-reperfusion injury is an vital factor which leads to the necrosis of the free skin flap.Moon JG reported that the regular clamp and release the vessel before the recovery of the vascular supply which could prevent the occurrence of reperfusion injury .But we find that the ischemic postconditioning of the skin flap can inhibit the apoptosis of the endothelial cell of the vessel,but the mechanism of the cell apoptosis is unclear.Bcl-2 is the key protein which can trigger the apoptosis of the mitochondrial pathway .The preliminary experiments suggest ①the ischemic postconditioning of the skin flap can activate the A2A receptor of the endothelia cell; ②the activation of the A2A receptor can prompt the expression of the Bcl-2,so the ischemic protection of the skin flap is mediated by the A2A receptor which can regulate the apoptosis of the mitochondrial pathway.We are to perform the ischemic postconditioning on the skin flaps and observe the expression of the endothelia cell and the apoptosis index etc al which can confirm the activation of the A2A receptor is the key factor which is against the apoptosis of the ischemic postconditioning on the overall level; to perform hypoxia postconditioning of the dermal hypoxia/ reoxygenation endothelial cell and observe the expression of the A2A receptor which can explore the character of the space and the time of the activation of the receptor;to regulate the apoptosis of the mitochondrial pathway on variable level and to observe the changes of the permeability of the mitochondrial membrane, the electric potential and the apoptosis gene which can illustrate the mechanism of the antiapoptosis of the endothelial cell mediated by the A2A receptor on the molecular level.The study will confirm that the activation of the endothelial A2A receptor is a key point leading to the ischemic protection of the skin flap,and explore the mechanmism of the antiapoptosis by the endothelial cell with the ischemic protection.

再灌注损伤是导致游离皮瓣坏死重要因素。Moon JG报道恢复皮瓣血供前规范性夹闭血管行缺血后处理可防止再灌注损伤。而我们发现对皮瓣行缺血后处理可抑制微血管内皮细胞凋亡，但机制不清。Bcl-2是启动线粒体凋亡途径的关键蛋白。预试验提示①皮瓣缺血后处理激活内皮细胞A2A受体；②A2A受体激活促进Bcl-2表达，因此我们假说皮瓣缺血后保护是由A2A受体介导而调控线粒体凋亡途径。拟对皮瓣再灌注损伤模型行缺血后处理，观察内皮损伤标志物、凋亡指数等，整体水平明确A2A受体激活是后处理抗凋亡关键；低氧/复氧皮肤内皮细胞模型行低氧后处理，观察A2A受体表达，细胞水平探讨受体激活的时空变化特点；不同层次调控线粒体凋亡通路，观察线粒体膜通透性、电位、凋亡基因变化，分子水平阐明A2A受体调控的抗内皮凋亡信号机制。研究将明确内皮A2A受体激活是对游离皮瓣行缺血后保护的关键靶点，揭示后处理内源性抗内皮细胞凋亡机理。

Moon JG报道恢复皮瓣血供前规范性夹闭血管行缺血后处理可防止再灌注损伤。而皮瓣再灌注损伤和微血管内皮细胞凋亡密切相关。Bcl-2是启动线粒体凋亡途径的关键蛋白。A2A受体激活调控Bcl-2表达。假说皮瓣缺血后保护是由A2A受体介导而调控线粒体凋亡途径。我们对新西兰大白兔皮瓣再灌注损伤模型行缺血后处理，首先建立了腹股沟皮瓣的再灌注损伤模型予以缺血4小时，再灌注8小时。进一步探讨缺血后处理的规范：联合对A2A受体进行激活和抑制，观察内皮损伤标志物、凋亡指数等，发现整体水平明确A2A受体激活是后处理抗凋亡关键；低氧/复氧皮肤内皮细胞模型行低氧后处理，观察A2A受体表达，细胞水平探讨受体激活的时空变化特点，A2A受体从细胞质外移动到细胞膜；不同层次调控线粒体凋亡通路，观察线粒体膜通透性、电位、凋亡基因变化，分子水平阐明A2A受体调控的抗内皮凋亡信号机制。研究明确内皮A2A受体激活是对游离皮瓣行缺血后保护的关键靶点，揭示后处理内源性抗内皮细胞凋亡机理。