基于“益气生脉、摄脉”理论的消渴通痹颗粒干预高糖诱导的EPCs-迁移-归巢及血管新生的分子机制研究

This essay aims to study the effects of Xiao Ke Tong Bi granules intervening on EPCs-migration -homing and angiogenesis with diabetic nerve hypoxic-ischemia for verifying the hypothesis that tonifying-Qi for activating-pulse and promoting blood circulation for dredging-collaterals or compound Chinese herbs may promote angiogenesis in model with diabetic peripheral neuropathy using transmission electro microscopy, immunohistochemistry, RT-PCR techniques, which base on the tonifying-Qi for activating and governing-pulse, tonifying-Qi for promoting granulation, promoting blood circulation for dredging collaterals and identity between venation system and blood vessel system classical theory. Furthermore, the proliferation, migration, homing and PI3K/AKt/eNOS signal pathway regulation of EPCs induced by Xiao Ke Tong Bi granules will be investigated for molecular mechanisms of angiogenesis and EPCs injury in vivo and in vitro using transwell assay, MTT assay, flow cytometry, which may provide scientific evidence and protein targets the entire for diabetic peripheral nerve injury.

本课题以中医的"益气-生脉"，"益气-摄脉"，"补气-生肌"，"活血-通络"等有关"气血关系"为指导，以"脉络-血管系统"同一性为出发点，从糖尿病神经缺血模型入手，采用超微电镜、免疫荧光、RT-PCR检测等手段研究益气活血中药消渴通痹颗粒对糖尿病神经缺血缺氧状态下内皮祖细胞（EPCs）迁移-归巢及新生血管形成的影响，验证"病证状态下'益气-生脉'、'活血-通络'法（中药）可促使缺血状态下血管新生"之假说；通过体外形态学、Transwell小室、MTT法及流式细胞仪等方法探讨益气活血中药含药血清对高糖诱导的内皮祖细胞保护、增殖、迁移及归巢等情况，并通过Western-blot检测Akt、p-Akt及eNOS、p-eNOS蛋白的表达，探究其对PI3K/Akt/eNOS信号通路的调控，揭示中药促血管生成及对内皮祖细胞保护的可能机制，为中医药防治糖尿病周围血管及神经病变提供科学实验依据及作用靶标。

本研究以中医“益气生脉、摄脉”，“活血通络”等“气血关系”为指导，以“脉络-血管系统”同一性为出发点，从糖尿病神经缺血模型入手，通过在体及体外研究益气活血中药消渴通痹颗粒对糖尿病神经缺血缺氧状态下内皮祖细胞（EPCs）迁移-归巢及新生血管形成的影响并探讨其可能分子机制，研究结果表明：益气活血中药能促进糖尿病大鼠周围神经新生血管形成，抑制血清炎症因子，升高糖尿病大鼠坐骨神经IgG、IgA灰度值；半定量RT-PCR检测缺血肢体肌肉中VGEF、HIF-1、PI3K\AKT\CREB1mRNA发现其在缺血性病变中迅速升高，促进血管内皮细胞增殖及血管生成效应；流式细胞检测发现其能促进糖尿病大鼠神经缺血状态EPCs-动员（迁移）；外周缺血神经紧密连接蛋白Occludin检测发现，益气活血中药有明显保护神经屏障作用；体外研究显示益气活血中药消渴通痹颗粒含药血清对高糖环境下EPCs有保护作用，并能促进EPCs增殖-迁移及官腔形成，Western-blot检测发现其可通过调控Akt、p-Akt及eNOS、p-eNOS蛋白表达，改善PI3K/Akt/eNOS分子信号通路，可能是其促血管生长的关键通路之一。