双向凝胶电泳技术在胰腺癌蛋白质组学研究中的应用及条件优化

Application and condition optimizing of two-dimensional gel electrophoresis in the study of pancreatic ductal adenocarcinoma proteomics

目的将双向凝胶电泳技术用于胰腺癌蛋白质组学研究并对其条件进行优化。方法对胰腺癌组织、癌旁组织、胰液等标本在组织研磨、裂解、蛋白抽提及双向凝胶电泳等各个环节进行调整和优化。结果液氮研磨法可得到更多的蛋白质点；超声方法去除组织中核酸效果较好；二硫苏糖醇（DTT）平衡时间12—15min，碘乙酰胺（IAA）平衡时间应等同或略长于DTT平衡时间时可得到高质量图谱；丙酮沉淀法可获得较多的胰液生物标志物蛋白点；将2—3根长度〈13cm的IPG胶条在1块凝胶上进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）的方法（Msoc）使双向电泳的重复性和通量显著提高。结论本研究成功建立了双向凝胶电泳技术平台，所获得的电泳图谱分辨率高、重复性好，能够较好地用于胰腺癌蛋白质组学研究。

Objective To put the two-dimensional （2D） gel electrophoresis （2-DE） into use for the study of pancreatic ductal adanocarcinoma（PDAC） proteomies, and to optimize the condition. Methods The samples of PDAC, normal tissue around PDAC and pancreatic juice were collected, and the sections including tissue grinding,lysising, proteins extraction and 2-DE were regulated and optimized. Results More proteins were obtained with grinding in liquid N,muleic acid disturbation in tisssues were cleared up more effectively by ultrasonic method. High quality electrophoresis pattern were got under the condition of 12-15 rains DTr equilibration time and IAA equilibration time of the similar or a little longer. More protein biomarker candidates in pancreatic juice sample were got by acetone precipitation. Repeatability and flux increased significandy after 2-3 IPG （ 〈 13 era） were loaded in one standard 2D gel at the same time during the SDS-PAGE. Conclusions An optimized two-dimensional gel electrophoresis system is set up successfully in this study, electrophoreais pattern of which shows high resolution and good repeatability, and can be used for the study of PDAC proteomics effectively.