儿童微小病变型肾病综合征尿蛋白组的双向电泳及PDQuest图像初步分析  

Preliminary analysis of urinary proteome of children minimal change nephrotic syndrome by two-dimensional gel electrophoresis and PDQuest software based image analysis

作　　者：黄艳军[1] 黄松明[1] 张爱华[1] 费莉[1] 徐颂周[1] 陈荣华[1] 

机构地区：[1]南京医科大学小儿肾脏病研究中心,江苏南京210029

出　　处：《第四军医大学学报》2006年第5期462-465,共4页Journal of the Fourth Military Medical University

基　　金：江苏省自然科学基金(BK2002030)

摘　　要：目的:建立儿童微小病变型肾病综合征尿蛋白组分析所需的固相pH梯度等电聚焦双向电泳及相关技术.方法:冷丙酮萃取肾病综合征患儿尿蛋白,去除白蛋白等高丰度蛋白、除盐,以固相IPGpH梯度等电聚焦为第一向,均一SDS胶聚丙烯酰胺凝胶(T=10%)为第二向,银染后PDQuest软件进行分析.结果:成功得到儿童微小病变型肾病综合征尿蛋白双向电泳图谱.尿蛋白用17cm,pH3-10L(线性)的IPG胶条进行二维电泳时上样量100μg,60000Vh的等电聚焦是恰当的.PDQuest7.3软件进行图像分析,检测到400个蛋白点.结论:用尿蛋白组学对儿童微小病变型肾病综合征的尿液进行研究是可行的.为今后从更高的水平来寻找肾脏疾病的功能蛋白和特异性蛋白质,阐明肾脏疾病发生发展的网络机制等后续研究提供了可能性.AIM： To analyze the urinary proteome of children minimal change nephrotic syndrome by the two-dimensional gel electrophoresis （2DGE） and PDQUEST software based image analysis. METHODS： The urinary protein of children nephrotic syndrome was isolated by cold acetone precipitation, treated to remove high abundance proteins by Aurum Serum Protein Kit and desalted by ReadyPrep^TM2-D Cleanup Kit. The rehydrated immobilized pH gradient （IPG） strips containing protein samples were isoelectric focusing （IEF）. After IEF, the equilibrated strips were transferred to the vertical SDS-polyacrylamide gel. The silver stained gels were scanned by UMAX PowerLook 1120 densitometer to acquire the digitalized images. Using new generation two dimensional image analysis software, PDQuest 7.3, the 2DGE of proteins extracted from urine was processed. The analysis procedure, including image acquirement, automated spot detection and quantification and analysis result report, was carried out. The method and the volume of loading sample, the choice of IPG gels, the concentration of SDS gels and the protocol for staining procedure were improved. RESULTS： Using the proper method stated above, satisfactory 2DGE maps of urinary protein were obtained and the preliminary analysis results were reported. The reasonable sample load and Voh-hours of urinary protein were about 100 μg and 60 000 Volt-hours for the 17 cm pH3-10 IPG strips. A total of 400 protein spots were visualized with PDQuest 7.3 software. CONCLUSION： Urinary proteomics can be applied in the study of urine protein of children minimal change nephrotic syndrome. We have used proteomic method to construct protein maps of urinary protein of children minimal change nephritic syndrome. At the level of proteome, we may use urinary proteomic techniques to find functional proteins and specific proteins （protein biomarker） of renal disease in the future and the technique will be further used in renal research to characterize network mechanisms of physiological processe

关 键 词：尿蛋白质组 双向电泳 图像分析 

分 类 号：R446.122[医药卫生—诊断学]