人肾小管上皮细胞蛋白质组的双向电泳及计算机图像分析  

Proteome research of Human renal tubular epithelial cell line HK-2 by two-dimensional gel electrophoresis and computer image analysis

作　　者：李娅[1] 陈楠[1] 王伟铭[1] 张群业[2] 黄秋花[2] 

机构地区：[1]上海第二医科大学附属瑞金医院肾脏科,200025 [2]上海第二医科大学附属瑞金医院上海血液研究所

出　　处：《中华肾脏病杂志》2003年第6期402-405,共4页Chinese Journal of Nephrology

摘　　要：目的 双向电泳(two-dimensional gel electrophoresis,2DGE)技术是蛋白质组研究中最为重要的蛋白质分离方法。建立并优化人肾小管上皮细胞蛋白质组分析所需的固相pH梯度等电聚焦双向电泳及相关技术。方法 由于不同的细胞系特殊性,样品处理亦不同,因此以人肾小管上皮细胞HK-2为研究对象,对样品液组成、样品处理、上样方式、上样量、IPG胶条和SDS-聚丙烯酰胺凝胶电泳染色方法等相关技术进行了研究和条件优化后,以固相pH梯度等电聚焦为第一向和SDS均一胶(T=12.5％)的水平电泳为第二向,研究人肾小管上皮细胞蛋白质组。结果 成功地得到了人肾小管上皮细胞双向电泳图谱,并通过ImageMaster 2D Elite 3.01软件进行图像分析,输出初步的检测结果。结论 建立蛋白质组研究的技术平台,为从更高的水平来寻找肾脏疾病的功能蛋白和特异性蛋白,阐明肾脏疾病发生发展的网络机制等后续研究工作奠定基础。Objective Two-dimensional gel electrophoresis(2DGE)is a key technique for proteomics. The aim of the research is to establish the two-dimensional gel electrophoresis and computer assisted image analysis for the proleome research of human renal tubular epithelial cell. Methods The human renal tubular epithelial cell line HK-2 was used as model. The rehydrated IPG strips containing protein samples were isoelectric focusing(IEF) . After IEF,the equilibrated strips were transferred to the vertical SDS gel. The silver stained gels were scanned by the densitometer,which generated the digitalized images. Using new generation two-dimensional image analysis software,ImageMaster 2D Elite 3.01 ,the 2DGE of proteins extracted from cultured renal tubular epithelial cell was processed. The analysis procedure,including image acquirement,automatic spot detection and quantification,analysis results report,was discussed. The method and volume of loading sample,choice of immobilized pH gradient(IPG)gels,concentration of SDS gels,preset of electric parameters,protocol for staining were improved. Results Using the proper method described above,satisfactory 2DGE maps of HK-2 was obtained and analysis results were reported. Conclusions We have used proteomic tools to construct protein maps of human renal tubular epithelial cell,the technique will be used in renal research to characterize physiological processes and disease.

关 键 词：肾小管 上皮细胞 蛋白质组 双向电泳 计算机图像分析 肾脏疾病 

分 类 号：R692[医药卫生—泌尿科学]