HGF-SPK-SIRT1信号通路在调控Ad-HGF干涉糖尿病微血管病变中的作用和机制

Diabetic microvascular diseases are the major complications,and also the main cause of death,disability for diabetic patients. Studies have shown that vascular dysfunction is an important reason for the occurrence and development of diabetic microvascular diseases, and endothelial dysfunction is a critical factor for vascular dysfunction.Endothelial progenitor cells (EPCs), can differentiate into endothelial cells(ECs), play a key role in repairing endothelial dysfunction. But in diabetes, high glucose induced reactive oxygen species (ROS) plays a key role in regulation of oxidative stress-induced apoptosis, senescence and dysfunction of EPCs. So, how to protect the EPCs damage induced by high glucose in diabetic patients to improve EPCs function and enhance vascular repair and angiogenesis capability, has become the critical problem of diabetic microvascular disease prevention..Our previous work showed that sphingosine kinase (SPK) activation mediated the biological function of hepatocyte growth factor (HGF), and SPK activation upregulates the expression of SIRT1. However, the roles of SPK-SIRT1 signal in endothelial cell protection remain unclear. .This project will observe the protective effects of Ad-HGF and the roles of SPK-SIRT1 in EPCs. The c-met/SPK/SIRT1 signal axis will be confirmed by lentivirus mediated RNA silencing of SPK. The roles and mechanism of SIRT1 in protecting EPCs from glucose injury will be investigated. We will generate the SIRT1 knockout mice to study the roles of SIRT1 in Ad-HGF biological function. The repair ability of SIRT1(wt) and SIRT1(-/-) bone marrow EPCs will checked by implantation into limb ischemia area in mouse models. We will further explore the corresponding molecular signals regulating mechanism of this gene drug, reveals the molecular mechanism of Ad-HGF resistance of stress, protection of EPCs apoptosis and senescence, improving EPCs artery vascular differentiation in high glucose stress environment. It is provide an important theoretical basis for the treatment of diabetic microvascular complications with the Ad-HGF gene drug, and also lay the foundation for its clinical application. Therefore, this project has important theoretical significance and guiding value.

糖尿病微血管病变是糖尿病致死、致残主要原因。高糖诱发的内皮祖细胞（EPCs）损伤及功能紊乱在其发生中起关键作用。我们前期结果提示SPK信号介导肝细胞生长因子（HGF）效应并上调SIRT1表达，但SPK-SIRT1通路是否发挥对EPCs保护作用并不清楚。本项目将研究Ad-HGF对高糖EPCs损伤保护，探其对SPK-SIRT1信号的激活作用；利用慢病毒介导shRNA分别干涉SPK 和SIRT1表达，确定HGF/SPK/SIRT1信号轴关联性，SIRT1 基因沉默对高糖诱导EPCs损伤保护及机制。繁殖SIRT1基因条件剔除小鼠，研究其EPCs特性及对Ad-HGF效应；通过给小鼠模型移植经Ad-HGF保护EPCs，观察移植SIRT1（WT）及SIRT1（-/-）骨髓EPCs参与血管修复情况。本项目将阐明HGF-SPK-SIRT1信号对内皮细胞保护及Ad-HGF作用机制，具有重要理论意义和实用价值。

背景与目的：高糖诱导的血管内皮细胞损伤是糖尿病血管并发症的始发因素和病理生理学基础，动物实验表明，携带肝细胞生长因子的重组腺病毒（Ad-HGF）对糖尿病血管损伤具有保护作用。本研究探讨Ad-HGF对高糖诱导的血管内皮细胞损伤的保护作用和机制。方法：分离、培养大鼠骨髓 MSCs，以最佳感染强度感染 Ad-HGF。建立2 型糖尿病大鼠急性后肢缺血模型，制备缺血模型后将 Ad-HGF 修饰的骨髓 MSC 于术后 10 min 内行手术部位多点肌内注射。手术注射后 6 周，取结扎后肢内与外侧肌肉组织，观察其组织病理学变化。利用酶切、回收、连接等方法构建了SPHK1干涉慢病毒载体，分别采用CCK8法、Transwell小室、流式细胞仪检测干涉SPHK1对人脐静脉内皮细胞（HUVEC）增殖、迁移、细胞周期和凋亡的影响，Matrigel小室检测细胞管状结构形成能力，PCR、WB检测对SIRT1、AKT、ERK、P38-MAPK通路蛋白表达的影响。检测转染Ad-HGF对高浓度葡萄糖（35mmol/L）诱导的HUVECs细胞存活率、NO产生、凋亡相关蛋白Bax/Bcl2、SPHK1、SIRT1、eNOS、MMP2蛋白表达的影响。结果： Ad-HGF 修饰的骨髓 MSCs 的局部应用可减轻糖尿病大鼠下肢缺血后肌肉组织损伤。干涉SPHK1基因可下调SIRT1、 AKT、ERK、P38-MAPK通路蛋白表达，显著降低HUVEC细胞增殖、迁移能力，增加细胞凋亡率。干涉SIRT1基因表达对SPHK1蛋白表达无影响。转染Ad-HGF可升高高糖诱导的HVECs细胞存活率，上调Bcl2、SIRT1、eNOS蛋白表达、下调Bax、MMP2蛋白表达。结论：Ad-HGF 修饰的骨髓 MSCs 对糖尿病大鼠下肢缺血性肌肉损伤具有明显的保护作用。在内皮细胞中，存在SPHK1/S1P-ERK/P38MAPK/AKT-SIRT1通路调节细胞增殖和迁移。转染Ad-HGF可通过激活内皮细胞SIRT1-eNOS通路、抑制细胞凋亡、增加细胞存活率，保护内皮细胞功能。