Netrin-1修饰的ASC对失神经支配后糖尿病小鼠血管新生的影响及分子机制

Diabetic peripheral nerve and vascular disease (DPNV) has high incidence, long course, and the early prevention and treatment are difficult. Adipose derived stem cells (ASC) play a pivotal role in promoting angiogenesis in diabetes, which shows a wide prospect of clinical application. However, our previous studies showed that ASC weakened in the target vessel parts of the survival, and differentiation capacity, which is the key to affect our active effects. As an important neurotrophic guiding factor, Netrin-1 not only takes part in the development of peripheral neuropathy and angiogenesis but also promotes endothelial cells and stem cells survival, migration, and blood vessel formation, however, its expression is lower in ASC. Thus we propose a hypothesis that overexpression of Netrin-1 by transfection in ASC might be better to promote its survival, migration and improve angiogenesis ability in diabetic mice after denervation. This study will based on our previous results, nuclear transfection will be adopted to make ASC overexpressing Netrin-1 and explore the influence of Netrin-1 on ASC survival, migration, and the differentiation capacity into endothelial cells. A unique diabetic mouse model of denervation will be established, and GFP gene will be used for tracing the roles of Netrin-1-ASC in promoting angiogenesis after cell transplantation. The numbers, diameters, and density of new vessels will also be analyzed at different transplantation times. Netrin-1 receptor DCC blocking agents and RNA Interference Silencing will be used to explore the possible effects of Netrin-1 transfected ASC in promoting angiogenesis in diabetic mouse model of denervation. The associated signal pathway will be discussed and the molecular mechanisms will be elucidated, which will bring new insights and theoretical basis into the prevention and treatment of diabetic peripheral nerve and vascular disease.

糖尿病周围神经血管病变(DPNV)发病率高、病程长、防治困难，血管合并神经病变是其难治愈的主要原因。脂肪干细胞(ASC)促进DPNV血管新生展示较好应用前景，但我们研究发现ASC在靶血管损伤部位存活及分化能力减低，是影响疗效的关键。作为重要的生长因子，Netrin-1可促进神经及血管损伤的修复，且对内皮细胞、干细胞具有保护作用。由此我们提出假设：通过使ASC过表达Netrin-1，可能会更好的促进失神经支配后糖尿病血管的新生。本项目拟在前期基础上，通过核转染使ASC过表达Netrin-1，体外检测其对高糖刺激下ASC存活、迁移、凋亡及分化的影响；体内移植治疗失神经支配糖尿病小鼠，检测新生血管血流、管径、密度变化，应用Netrin-1相关受体阻断剂及siRNA技术探讨相关信号通路的变化，阐明Netrin-1修饰的ASC促进失神经支配后血管新生的分子机制，为DPNV的防治提供新思路。

本项目旨在探索Netrin-1对高糖刺激下ASC存活、增殖、凋亡及功能的影响，Netrin-1修饰的ASC对失神经支配后糖尿病小鼠血管新生的作用及相应的其分子机制。 . 首先，我们验证了Netrin-1对高糖刺激下ASC增殖、迁移、粘附、凋亡及功能的影响。我们利用核转染技术，建立一套稳定的使ASC 过表达Netrin-1的体系，明确核转染Netrin-1的效率及Netrin-1表达持续时间；我们通过CCK8实验发现Netrin-1修饰的ASC的增殖能力明显更强，利用Transwell实验证实Netrin-1可以促进ASC的迁移能力，粘附实验发现Netrin-1可以提高ASC的粘附能力，流式细胞术发现Netrin-1修饰的ASC在高糖条件下的凋亡率明显更低；ELisa发现在高糖条件下Netrin-1修饰的ASC分泌的VEGF, b-FGF, HGF, TNF-α, PDGF,EGF, IGF-1 和Netrin-1显著更高。. 其次，我们探索了Netrin-1修饰的ASC对失神经支配后糖尿病小鼠的血管新生的作用。我们建立糖尿病小鼠的失神经模型，局部分别注射PBS、ASC和Netrin-1修饰的ASC，我们通过激光多普勒检查发现注射Netrin-1修饰的ASC的糖尿病小鼠有着显著更高的激光多普勒灌注指数，我们通过免疫荧光和免疫组化也证实了Netrin-1修饰的ASC有着显著更多的新生血管。. 最后，我们揭示了Netrin-1促进ASC存活、分化及促进失神经支配后糖尿病小鼠血管新生的分子机制。我们通过Western blot发现，不论在体外还是体内实验中，Netrin-1修饰的ASC中PI3K/AKT/eNOS/P-38/NF-κB信号通路的磷酸化表达量均明显上调。该研究结果表明Netrin-1通过PI3K/AKT/eNOS/P-38/NF-κB通路促进ASC的存活、分化及失神经支配后糖尿病小鼠的血管新生。. 我们的研究结果阐明了Netrin-1可调控ASC的存活、增殖、凋亡及促进血管新生的作用及分子机制，该研究结果有希望用于糖尿病周围神经血管病变的治疗。