维生素D受体在细胞核水平拮抗糖尿病肾病炎症反应的机制

项目名称： 维生素D受体在细胞核水平拮抗糖尿病肾病炎症反应的机制

项目编号： No.81470961

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 张浩

作者单位： 中南大学

项目金额： 73万元

中文摘要： 炎症在糖尿病肾病(DN)发生发展中起重要作用，VDR通过抗炎发挥肾保护作用，但其机制尚未完全阐明。我们前期研究发现2型DN患者存在核内VDR下调，且与NF-κB、MCP-1等促炎因子呈负相关，细胞研究发现VDR和NF-κB均能与核孔蛋白214(Nup214)结合，且在高糖刺激下呈现核内外差异性表达。本研究拟构建不同片段VDR、NF-κB、Smad2和Nup214高表达质粒以及siRNA质粒，分别转染高糖诱导的足细胞/系膜细胞/肾小管上皮细胞，采用CoIP等方法证实VDR可竞争性抑制NF-κB、Smad2与Nup214结合及入核转运，采用ChIP、EMSA等方法证实VDR可通过特异性识别抑炎因子MIP-1β、PTPN2启动子上的维生素D反应元件调控转录；拟在db/db和STZ诱导糖尿病小鼠及VDR-/-小鼠模型上证实VDR在胞核水平拮抗DN炎症的分子机制，为早期防治DN提供新线索和实验支持。

中文关键词： 糖尿病肾病；维生素D核受体；核孔蛋白214；入核转运；转录调控

英文摘要： Inflammation plays an important role in the pathegenesis and progression of diabetic nephropathy (DN), and vitamin D receptor (VDR) is widely recognized as a key reno-protective factor through its complex anti-inflammatory effects. Our previous clinical research has found significant down-regulation of nuclear VDR expression in type 2 DN patients, which is negatively associated with the expressions of several pro-inflammatory factors, including NF-κB and MCP-1. Our preliminary in vitro studies have further demonstrated that both VDR and NF-κB bind to nuclear pore proteins 214 (Nup214) for nuclear transportation, and that either VDR or NF-κB has shown differential nuclear or cytoplasmic distributions under high glucose conditions. Here, we intend to establish three high glucose-stimulated cell line models, including podocyte，mesangial and renal tubular epithelial cells, transiently transfect these cell models with epitope-tagged full-length, N-terminal, C-terminal constructs or siRNAs of VDR, NF-κB, Smad2 or Nup214, and investigate with CoIP whether VDR could competitively inhibit Nup214 binding of NF-κB or Smad2 and their subsequent nuclear transportion; we will use ChIP and EMSA assays to evaluate whether VDR, as a nuclear transcription factor, specifically recognizes and binds to putative vitamin D response elements on the promoter of two anti-inflammatory factors, MIP-1β and PTPN2, and regulates their transcriptions; we will further explore novel molecular mechanisms involved in the anti-inflammatory actions of nuclear VDR with STZ-induced diabetic mice and VDR-/- mice based on our in vitro findings in order to provide new clues and evidence basis for the prevention and treatment of DN.

英文关键词： Diabetic Nephropathy;Nuclear Vitamin D Receptor;Nucleoporin 214;Nuclear Import;Transcription Regulation