MitoK+ATP/CXCR4/Rho GTPase通路在EPCs移植促进糖尿病大鼠动脉损伤后再内皮化中的作用及机制研究

PCI is an important treatment of coronary heart disease. Clinical and basic experiments found that individuals with diabetes are more likely to have delayed re-endothelialization, stent thrombosis and restenosis than non-diabetic individuals. Transplantation of EPCs can promote repair of damaged vascular endothelium, but the number and function of EPCs in diabetic individuals are both impaired. Therefore, it is of great significance to study the drug pretreatment before transplanting EPCs to promote the repairing of damaged vascular endothelium after PCI. The applicant's preliminary study found that after oral administration of nicorandil, opening mitochondrial K + ATP channel in diabetic rats can inhibit neointimal hyperplasia after balloon injury. Oral administration of nicorandil opens mitochondrial K + ATP channels to promote re-endothelialization following balloon injury in SD rats. 100μM Nicorandil promoted the proliferation and migration of EPCs and up-regulated the expression of CXCR4 in EPCs, which was inhibited by 5-HD, a specific inhibitor of mitochondrial K + ATP channel. 100μM nicorandil can promote Rho GTPase expression by upregulating CXCR4 in EPCs. We aim to verify the hypothesis that pre-treatment EPCs with nicorandil, a mitochondrial K + ATP-opener, before transplantation can inhibit their apoptosis and senescence and promote their homing, and therefore promote re-endothelialization in diabetic rats. We also aim to clarify the mechanism of mitochondrial K + ATP / CXCR4 / Rho GTPase pathway in transplanting EPCs to promote re-endothelialization.

PCI术是冠心病的重要治疗手段。然而，合并糖尿病的个体较非糖尿病个体PCI术后再内皮化进程显著延迟。移植EPCs可促进修复损伤的血管内皮，但糖尿病个体EPCs的数量及功能均受损。开展移植EPCs前药物预处理促进糖尿病个体PCI术后再内皮化的研究具有重要意义。申请人前期研究发现大鼠动脉损伤后，口服尼可地尔开放线粒体K+ATP通道可促进再内皮化。体外实验发现尼可地尔可促进被高糖抑制的EPCs增殖和迁移，并上调CXCR4表达，而该促进作用可被5-HD阻断。尼可地尔可通过上调EPCs内CXCR4来促进Rho GTPase的表达。课题拟从动物、细胞、分子水平揭示糖尿病大鼠动脉球囊损伤后，线粒体K+ATP开放剂尼可尔在EPCs移植前预处理，抑制其凋亡和衰老，促进其归巢，从而促进再内皮化的作用；并阐明线粒体K+ATP/CXCR4/Rho GTPase通路在移植EPCs促进再内皮化中的作用和机制。

背景：合并糖尿病的个体较非糖尿病个体PCI术后再内皮化进程显著延迟。移植EPCs可促进修复损伤的血管内皮，但糖尿病个体EPCs的数量及功能均受损。尼可地尔开放线粒体K+ATP通道可促进再内皮化，可促进被高糖抑制的EPCs增殖和迁移，并上调CXCR4表达，而该促进作用可被5-HD阻断。课题拟从动物、细胞、分子水平揭示糖尿病大鼠动脉球囊损伤后，线粒体K+ATP开放剂尼可尔在EPCs移植前预处理，抑制其凋亡和衰老，促进其归巢，从而促进再内皮化的作用；并阐明线粒体K+ATP/CXCR4/Rho GTPase通路在移植EPCs促进再内皮化中的作用和机制。.内容：培养大鼠骨髓源EPCs，建立大鼠颈总动脉球囊拉伤模型，移植PKH26标记的EPCs。在拉伤后第1天、第4天、第14天取材, 分析EPCc粘附、内皮修复和内膜增生。分离ZDF糖尿病大鼠骨髓源EPCs，构建 ZDF 糖尿病大鼠模型，颈动脉球囊损伤后，移植经尼可地尔、线粒体K+ATP 通道阻断剂 5-HD、CXCR4 阻断剂 AMD3100、Rho GTPase 阻断剂 CCG1423 等不同预处理并行 PKH26 标记的 EPCs。通过免疫组化、流式细胞术、共聚焦显微镜、western blot 等技术，明确尼可地尔预处理可促进 EPCs 在血管损伤面归巢并促进再内皮化进程的作用。在动物水平，明确移植尼可地尔预处理的EPCs 对糖尿病大鼠动脉损伤后再内皮化的促进作用，并探讨线粒体K+ATP/CXCR4/Rho GTPase 信号通路中关键分子的变化。.结果：移植EPCs在球囊拉伤后1天趋化黏附到损伤的血管表面。4天损伤血管管腔表面的EPCs明显减少。14天移植EPCs减少新生内膜增生，但覆盖的内皮未见PKH26荧光。EPCs分泌VEGF和IGF-1，移植后大鼠血清中VEGF和IGF-1显著增加。提示移植EPCs抑制内膜增生主要机制为旁分泌。线粒体K+ATP/CXCR4/Rho GTPase信号通路影响了糖尿病大鼠EPCs凋亡、增殖、衰老及旁分泌，从而促进了内皮修复并抑制了内膜增生。.结论：移植EPC可以趋化黏附到损伤血管表面，促进血管内皮修复并抑制内膜增生，其主要机制是通过旁分泌而非直接分化为内皮细胞。线粒体K+ATP/CXCR4/Rho GTPase信号通路影响EPCs凋亡、增殖、衰老及旁分泌，抑制了内膜过度增生。