靶向抑制肾脏microRNA-503和microRNA-181d表达治疗KKAy小鼠糖尿病肾病的机制研究

miRNA功能的调控为糖尿病肾病（DN）治疗提供了新靶点。本研究拟在前期2型糖尿病KKAy小鼠肾小球蛋白和miRNA表达谱研究的基础上，选择在KKAy小鼠肾小球内表达明显上调且其异常表达可被氯沙坦抑制的miRNA-503和miRNA-181d，应用超声微泡技术，分别向KKAy小鼠肾脏转染miRNA-503和miRNA-181d的敲除质粒，观察其对DN的治疗作用；体外培养肾小球系膜细胞、内皮细胞和足细胞，分别转染miRNA-503和miRNA181d的过表达和敲除质粒，观察其对高糖培养的3种肾脏固有细胞损伤、凋亡、增殖能力的影响。应用蛋白组学技术，检测转染后小鼠磁珠分离肾小球和3种固有细胞蛋白表达谱的变化，进行GRP75、Wnt3a、PI3K-AKT、BCL-2等关键靶基因的功能分析。旨在明确miRNA-503和miRNA-181d在DN发病中的网络式调控作用，为DN的诊治提供新思路和新靶点

Restoring or inhibition expression of miRNAs provide a new approach to management of diabetic nephropathy(DN). Our previous proteomics and microarray studies suggested that the expression of miRNA-503 and miRNA-181d were significantly up-regulated in the glomeruli of KKAy mice and inhibited by losartan treatment. In present study, miRNA-503 knockout or miRNA-181d knockout plasmids will be delivered into the living kidney of diabetic KKAy mice by ultrasound-microbubble-mediated gene transfer, and the renoprotective role will be observed. By in vitro study, glomerular mesangial cells, endothelial cells and podocyte will be transfected by miRNA-503 and miRNA-181d mimic or knockout plasmids respectively. Apoptosis,injury and proliferation assay of renal intrinsic cells cultured under high glucose will be conducted. The effect of miRNA-503 and miRNA181d on protein expression profiles of KKAy mice glomeruli isolated by Dynabeads and intrinsic cells under high glucose will be investigated using 2D-DIGE and MALDI-TOF mass spectrometry.The expression and function of predicted target genes of miRNA-503 and microRNA-181d including GRP75, Wnt3a, PI3K, AKT and BCL-2 will be observed. The objective is to identify the regulatory networks and the crosstalk of miRNA-503 and miRNA-181d and to explore novel therapeutic targets for DN.