利用斑马鱼肾脏损伤模型研究GADD45B和APLP1基因对足细胞损伤的影响及分子机制

项目名称： 利用斑马鱼肾脏损伤模型研究GADD45B和APLP1基因对足细胞损伤的影响及分子机制

项目编号： No.81470943

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： 陈朝红

作者单位： 中国人民解放军东部战区总医院

项目金额： 73万元

中文摘要： 足细胞损伤是肾小球疾病发生发展的关键环节。课题组前期利用TGFbr1(AAD)和Dicer fl/fl:NPHS2-Cre两种足细胞损伤相关肾小球疾病模型，筛选发现一组一致性变化的差异表达基因，其中上调基因GADD45B和APLP1在病例对照研究中进一步得到证实,提示其可能与肾小球疾病的发生发展高度相关。本项目以GADD45B和APLP1为靶基因，利用斑马鱼模型技术平台，结合通路克隆、Uas/Gal4和CRISP/Cas9等系统，分别构建靶基因在足细胞上特异性过表达和基因敲除的斑马鱼模型；分析特异性提高或抑制靶基因表达对肾脏组织形态、结构和功能的影响；体内外研究相结合，系统比较靶基因过表达或抑制对凋亡信号通路和足细胞损伤基因甲基化的影响，解析靶基因与足细胞损伤发生、发展的关系及其分子基础；对筛选到的候选足细胞损伤标志物进行临床验证和转化评估。研究结果对深入阐明肾小球疾病分子机制有积极意义。

中文关键词： 足细胞损伤；生长抑制与DNA损伤基因45B；凋亡；甲基化；斑马鱼

英文摘要： It is well known that podocyte injury is an early event leading to glomerular disease. However, the molecular mechanism of early podocyte injury in glomerular disease remains unclear. In order to better understand the pathogenesis of early podocyte injury in glomerular disease, two mouse models of glomerular disease were generated and characterized : 1) TGFbr1(AAD):NPHS2-rtta (by crossing transgenic mouse line of constitutively active TGF-beta receptor type 1 (TGFbr1(AAD) into the NPHS2-rtta transgenic line and 2) Dicer f/f:NPHS2-Cre. To identify the common genes that may be responsible for the similar phenotypes of the two models, we performed microarray analyses of the glomeruli using both mouse models. 20 genes were identified to be regulated similarly in these two glomerular disease models. Next we determined if those genes identified in mouse glomerular disease models are clinically relevant. Through real-time RT-PCR analysis with laser micro dissected glomeruli and histological studies, we identified growth arrest and DNA damage gene 45B (GADD45B) and amyloid like protein gene (APLP1) were regulated in the same way as in the animal models. The conservative regulation of the genes is highly suggestive of that they play a critical role in the development of glomerular diseases. In this proposed project, we will be conducting functional studies on the genes by utilizing zebra fish as in vivo animal model. Pod::NTR-mCherry transgenic fish or puromycin nucleotide microinjection fish will be used as inducible podocyte injury model. MultiSite gateway strategy and Uas/Gal4 system will be used for target gene expression and podocyte specific transgenic animal model construction. In addition, CRISP/Cas9 will be used to knockdown gene expression. We will be assessing the morphological and functional changes in the kidney with GADD45B and APLP1 over-expression/knock-down through in situ hybridization, immunofluorescence, electron microscopy and proteinuria detection via GFP-tagged vitamin D-binding protein transgenic fish. The effect of GADD45B and APLP1 overexpression/know-down on the apoptosis and methylated profiles in podocytes will be obtained and analyzed to undermine the mechanism of GADD45B and APLP1 on podocyte injury.The identified molecular related with podocyte injury will be validated for diagnostice biomarker in glomerulare disease. The project will highlight the mechanism of podocyte injury and glomerular disease.

英文关键词： podocyte injury;GADD45B;apoptosis;methylation;zebrafish