Netrin-1对糖尿病肾病血管新生的调节作用及机制

Angiogenesis plays an important role in the early stage of diabetic nephropathy.The new born vascular is immature and the high permeability of it leading to albumin extravasation, which causes related pathological damage to kidney . The axon-directed factor Netrin-1 is a secreted protein which has multi-function.The Netrin-1 and its receptors UNC5B and neogenin are found in a number of tissues and organs, playing a role in regulating angiogenesis. However, the regulation of neovascularization in kidney by Netrin-1 has never been studied. Studies have been shown that Netrin-1 has a protective effect in diabetic nephropathy, but its specific mechanism is unclear. Our preliminary study has been shown that Netrin-1 is elevated in the urine of DN patients, and the extracorporal experiments demonstrate that Netrin-1 can inhibit the migration and formation of human glomerular endothelial cells, and its receptor neogenin is highly expressed in glomerular endothelial cell. In this study, we construct specific neogenin knockout model mice by using cell biology and cre-loxp technique, exploring the mechanism of Netrin-1 / neogenin to angiogenesis of diabetic nephropathy. Further more, we want to detect the recombinant protein Nerin-1 as a perspective treatment and prevention to diabetic nephropathy in pharmacodynamics ，so as to provide new ideas and methods for Diabetes nephropathy.At the same time it can provide an important theoretical basis and experimental basis to the development of recombinant protein drugs.

血管新生在糖尿病肾病（DN）早期发挥重要作用，新生不成熟血管通透性增加致白蛋白外渗，引起肾脏病理损伤。轴突导向因子Netrin-1是一种多功能分泌型蛋白，发现Netrin-1与其受体UNC5B及neogenin在多个组织器官发挥调节血管新生作用；但Netrin-1对肾脏血管新生的调节从未研究。已有研究证明Netrin-1在DN中起保护作用，但机制不清。前期研究发现Netrin-1在DN患者尿液中升高，体外Netrin-1可抑制人肾小球内皮细胞的迁移、成管作用，neogenin在肾小球内皮细胞高表达。本课题运用细胞生物学及cre-loxp技术构建特异性内皮细胞neogenin基因敲除模型鼠，探讨Netrin-1／neogenin对DN血管新生作用的机制，并进一步对重组蛋白Netrin-1作为DN的治疗手段进行初步的药效学研究，为DN防治提供新思路及方法，为重组蛋白治疗提供理论依据及实验基础。

完成了Netrin-1调节糖尿病肾病血管新生中的相关研究。神经导向因子 Netrin-1、UNC5B不仅在神经轴突发育并且可以在血管发生中起重要作用。本课题组通过细胞实验和分子生物学实验验证 Netrin-1在DN组尿液中明显高于对照组，Unc5b在人及大鼠肾脏都有表达，并且主要分布在肾小球的血管内皮细胞；DN组Unc5b表达明显多于正常对照组；沉默UNC5B表达后适合浓度外源Netrin-1因子可以抑制HRGECs迁移和成管作用，说明UNC5B和其配体Netrin-1共同作用，调节DN血管新生作用。这种作用很可能是通过抑制可抑制VEGFA/VEGFR2下游Src的磷酸化发挥作用。抑制肾脏UNC5B表达同时增加外源Netrin-1可能成为抑制DN中肾脏血管新生的有效方法，从而减慢肾脏损害进度 。. 同时在对Netrin-1研究样本搜集和检测过程中，建立了肾脏病临床医学研究中心及DN样本库，并检测发现Netrin-1在成人心外术后发生急性肾损伤（AKI）具有一定诊断价值。建立了db/db糖尿病肾病小鼠模型，及 STZ诱导SD大鼠的糖尿病肾病模型，并通过检测肾脏蛋白质组学建立了DN琥珀酰化蛋白修饰组学数据库及DN肾脏肾小球磷酸化蛋白数据库。为糖尿病肾病临床及科研提供新的思路。