人胆汁蛋白质组双向电泳图谱的建立与初步分析  

Establishment and preliminary analysis of a 2-D human biliary map

作　　者：陈波[1] 郑建伟[1] 王剑明[1] 田俊[2] 王淳本[2] 邹声泉[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外科,武汉市430030 [2]华中科技大学同济医学院蛋白质组学研究中心,武汉市430030

出　　处：《中华肝胆外科杂志》2007年第8期536-539,共4页Chinese Journal of Hepatobiliary Surgery

基　　金：国家高新技术研究发展计划(863)项目(编号:2002AA241061)

摘　　要：目的建立人胆汁蛋白质组双向电泳图谱,用于胆汁比较蛋白质组学研究。方法A、B两例胆汁标本分别取自于一例肝门部胆管癌及一例胆总管结石病人。标本经脱盐、脱脂、去除核酸纯化处理后,分别各取350μg以固相pH梯度等电聚焦(IEF)为第一向,SDS-PAGE垂直电泳为第二向进行双向电泳(2-DE)。应用图像分析软件对银离子染色的2-DE图谱进行图像分析。结果经纯化处理的胆汁样本双向电泳图谱中的各个蛋白质点分离清晰,无明显的横向及纵向拖尾;A、B两例胆汁样本2-DE图谱中分别分离出250和216个蛋白质点。其中仪在A例出现的有187个,仅在B例出现的有153个;A、B两例均出现但表达量存在明显差异的有63个。结论本实验建立了一种较为理想的人胆汁蛋白质纯化制备及双向电泳技术;通过对良、恶性阻塞性黄疸胆汁2-DE图谱的匹配分析发现了大量的差异蛋白质点,为开展胆汁比较蛋白质组学研究筛选胆道疾病相关标志物奠定了前期实验基础。Objective To establish a 2-D human biliary map for the purpose of comparative proteomie analysis. Methods The bile was obtained by T tube 3 d of external drainage from 2 patients with obstructive jaundice due to eholangioeareinoma and eholelithiasis, respectively. The unfraetionated bile received sonieation and ultraeentrifugation to remove the cellular debris and nucleic acid as a crude separation. For further purification, the commercial kits CleanaseiteTM and MICROCON were adopted to remove the abundant lipid and bile salt. After quantified by BCA, each purified sample of 350 μg was run through immobilized pH gradient （IPG, pH3-10, 18cm） isoeleetrie focusing electrophoresis as the first dimension, and then run through vertical SDS-PAGE eleetrophoresis as the second dimension. The 2 DE maps were visualized after silver staining and analyzed with Image Master 2D software. Results Common gel artifacts such as streaking and smearing could be reduced by a general clean up of the bile samples prior to isoeleetrie focusing （IEF）. There were 250 and 216 spots obtained in 2-DE maps of sample A and sample B, respectively, of which 63 matched spots increased or decreased in quantity. Other 187 and 153 spots were exclusively found in eholangioeareinoma and eholelithiasis group, respectively. Conclusions A reliable sample preparation strategy is developed and an optimized 2-D human biliary map established in this study. The differentially displayed proteome be tween the pathological bile obtained from benign and malignant bile duct obstruction indicates the potential application of bile to identify the biomarkers for biliary tract pathological changes by means of comparative proteomies.

关 键 词：胆汁 蛋白质组 双向电泳 比较蛋白质组学 

分 类 号：R657.4[医药卫生—外科学]