激光捕获显微切割技术结合^(18)O标记定量蛋白质组技术在胃癌标志物筛查中的应用研究  

Quantitative Proteome Analysis of Clinical Gastric Adenocarcinoma Using ^(18)O Stable Isotope Labeling and LCM

作　　者：张志强[1,2] 李茂玉[3] 张桂英[1] 彭芳[3] 姚慧欣[3] 李美香[3] 肖志强[3] 陈主初[3,4] 

机构地区：[1]中南大学湘雅医院消化科,长沙410008 [2]新疆医科大学第一附属医院消化科,乌鲁木齐830054 [3]中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室,长沙410078 [4]中南大学湘雅医学院肿瘤研究所,长沙410008

出　　处：《生物化学与生物物理进展》2009年第3期311-322,共12页Progress In Biochemistry and Biophysics

基　　金：中南大学博士点基金资助项目(0575240);湖南省科技重大专项资助(04sk1006-2)~~

摘　　要：建立一种更加精确地分离鉴定胃癌特异肿瘤标志物的定量蛋白质组学技术.首先采用激光捕获显微切割技术(LCM)纯化胃腺癌细胞及胃黏膜良性上皮细胞,将裂解的样本总蛋白经过1DSDS-PAGE预分离,然后采用18O/16O分别标记两种样本酶切后的多肽混合物.结合纳升级液相色谱(Nano-HPLC-MS/MS)定量地鉴定胃癌细胞和胃黏膜良性上皮细胞的差异表达蛋白.共筛选出78个差异表达蛋白,其中42个蛋白质在胃癌组织中表达上调,36个蛋白质下调.Western blot技术验证了其中几个差异蛋白(moesin,periostin,annexin A2,annexin A4)的表达,与蛋白质组学研究的结果一致.LCM技术结合18O稳定同位素标记的定量蛋白质组学技术,为研究胃癌发生机制、筛选胃癌的分子标志物提供了新的思路,亦为诸如胃癌等复杂体系蛋白质的分离鉴定提供了新的技术选择.A high accuracy method for quantitative expression proteomics was developed for the identification of putative markers in gastric adenocarcinoma. Firstly, gastric adenocarcinoma and nonmalignant epithelial cells were obtained through laser capture microdissection （LCM）, which were proteolysis and then prefractionated by 1D SDS-PAGE. The post-digestion 180/t60 labeling method followed by Nano-HPLC-MS/MS were conducted to identify the differentially expressed proteins between gastric adenocaicinoma and nonmalignant gastric mucosa. A total of 78 differential proteins were identified, among these proteins, 42 proteins are over-expressed in gastric adenocarcinoma tissues and 36 proteins are down-expressed. Some of these differentially expressed proteins （moesin, periostin, annexin A2, annexin A4） were further confirmed by Western blot analysis. The quantitative proteomic protocol of ^18O stable isotope labeling coupled with LCM was an effectively alternate technique for complicated proteins such as gastric cancer.

关 键 词：胃癌 激光捕获显微切割 定量蛋白质组学 18O标记 

分 类 号：R57[医药卫生—消化系统]