双向电泳技术在筛选膀胱癌特异标志蛋白中的应用  

Two-dimensional electrophoresis for the screening of biomarker in carcinomas of the urinary bladder

作　　者：靳胜[1] 张曼[1] 张秀敏[1] 许华林[1] 张敏[1] 郝林[1] 

机构地区：[1]北京世纪坛医院临床检验中心,100038

出　　处：《中华检验医学杂志》2006年第5期445-448,共4页Chinese Journal of Laboratory Medicine

摘　　要：目的建立高分辨率的膀胱移行上皮癌不同时期的双向凝胶电泳图谱,并初步分析其蛋白质表达情况,寻找其差异表达蛋白,为寻找癌变相关蛋白奠定实验基础。方法用固相pH梯度双向凝胶电泳技术分离膀胱移行上皮癌不同分期的组织,凝胶考马斯亮蓝染色,然后利用GS-800Calibrated Densitometer凝胶成像系统获取图像,用PDQuest2D分析软件对图像进行背景消减、斑点检测、匹配、斑点位置重复性的系统分析,寻找差异表达蛋白。在此基础上应用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)得到相应的肽质指纹图谱,然后搜索数据库鉴定部分差异蛋白质。结果获得了背景清晰、分辨率和重复性较好的双向凝胶电泳图谱,三块胶的平均蛋白质匹配率达76%,蛋白质点在位置上有较好的重复性,不同胶间的蛋白质点在等电聚焦IEF方向偏差是(0·98±0·26)mm,SDS-PAGE方向偏差是(1·23±0·22)mm,并且得到了若干与细胞增生、分化、周期调控信号转导或肿瘤发生有关的蛋白质,例如SFN,热休克蛋白-27等。结论为进一步探讨癌变机理及筛选其特异性分子标志物打下方法学基础。Objective This study was designed to establish and optimize the two-dimensional gel electrophoresis （2-DE） for the proteome analysis of human Transitional Cell Carcinomas of the Urinary Bladder , and to established the two-dimensional gel electrophoresis （2-DE） profile of human Transitional Cell Carcinomas of the Urinary Bladder（TCC） of different stages in carcinogenic process. The basic aim is to perform differential analysis and provide a basis for identifying carcinogenesis-associated protein of TCC. Methods After obtaining samples of the normal, invasive tissue, noninvasive tissue, a series of methods Such as sample preparation, electrophoresis parameters, gel concentration, PDQuest 2DE analysis software, peptide mass fingerprint （PMF, MALDI-TOF-MS）were used to analyses these tissues. Results The good 2DE patterns of different tissues including resolution and reproducibility were obtained. After coomassie Brilliant Blue R-250 staining, the average matching ratio was 76%. There was a good reproducibility of spot position in 2-DE map, with average deviation in IEF direction of （0. 98 ± 0. 26） mm, while in SDS-PAGE direction it was （ 1.23 ± 0.22） mm. The 2-DE patterns with good quality had been obtained. Ten protein spots chosen randomly from different stages were identified by PMF, some of which were involved in the cell proliferation, differentiation, cycle regulation, and tumor occurrence, such as Heat shock protein-27, SFN etc. Conclusions The results provide a fundamental basis for further study of mechanism of human Transitional Cell Carcinomas of the Urinary Bladder and screening its specific marker.

关 键 词：电泳 疑胶 双向 膀胱肿瘤 蛋白质组 

分 类 号：R737.14[医药卫生—肿瘤]