应用激光捕获显微切割和蛋白质组学技术筛选鼻咽癌的差异表达蛋白质  

Screening for differentially expressed proteins in nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis

作　　者：易红[1] 程爱兰[1] 黄卫国[1] 张鹏飞[1] 李茂玉[1] 彭芳[1] 李峰[1] 李萃[1] 陈主初[1] 肖志强[1] 

机构地区：[1]中南大学湘雅医院医学实验研究中心,长沙410008

出　　处：《中南大学学报（医学版）》2008年第5期375-383,共9页Journal of Central South University ：Medical Science

基　　金：国家重点基础研究发展规划项目(973)(2001CB5120);芙蓉学者特聘教授科研奖励基金(湘教通[2007]362号);教育部跨世纪优秀人才培养计划基金(教技函[2002]48号);湖南省科技厅重大科技专项(06SK2004)~~

摘　　要：目的:筛选鼻咽癌(nasopharyngeal carcinoma,NPC)的差异表达蛋白质,为寻找NPC的分子标志物提供科学依据。方法:采用激光捕获显微切割技术(laser capture microdissection,LCM)分别从NPC组织和正常鼻咽上皮组织(normal nasopharyngeal epithelial tissue,NNET)中纯化NPC细胞和正常鼻咽上皮细胞(normal nasopharyngeal epithelial cells,NNEC),应用二维凝胶电泳(2-DE)分离LCM纯化细胞的蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和电喷雾电离串联质谱(ESI-Q-TOF-MS)鉴定差异蛋白质点,Western印迹和组织芯片(tissue microarray,TMA)免疫组织化学验证差异蛋白鳞状细胞癌抗原1(SCCA1)在两种组织中的表达水平。结果:建立了LCM纯化的NPC细胞和NNEC的2-DE图谱,质谱鉴定了36个差异蛋白质,其中20个蛋白只在NPC表达或表达上调,16个蛋白在NPC中表达下调或缺失。Western印迹和组织芯片免疫组织化学结果显示:SCCA1在NPC中的表达水平较NNET明显下调,与比较蛋白质组学结果一致。结论:36个差异蛋白可能与NPC的发病有关,为筛选NPC分子标志物奠定了基础。Objective To search for the differentially expressed proteins of nasopharyngeal carcinoma （NPC）, and provide scientific evidence for identifying molecular biomarkers for NPC. Methods Laser capture microdissection （LCM） was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues （NNET）. Two-dimensional gel electrophoresis （2-DE） was used to separate the total proteins of microdissected NPC and NNET, PDQuest software was applied to analyze 2-DE images, and the differential proteins between the 2 types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Western blot and immunohistochemistry of tissue microarray were used to detect the expression of the differential protein SCCA 1 in NPC and NNET. Resuits 2-DE patterns of microdissected NPC and NNEC were established, and 36 differential proteins in the NPC and NNEC were identified,20 of which only expressed or up-regulated in NPC and 16 only expressed or up-regulated in NNET. The differentially expressed level of SCCA1 in the NPC and NNET was confirmed by Western blot and immunohistochemistry of tissue microarray. Conclusion Thirty-six differentially expressed proteins identified in this study may be associated with the carcinogenesis of NPC, and may be candidate molecular biomarkers for NPC.

关 键 词：鼻咽癌 激光捕获显微切割技术 蛋白质组学 二维凝胶电泳 基质辅助激光解吸电离飞行时间质谱 电喷雾电离串联质谱 免疫印迹 组织芯片 

分 类 号：R739.63[医药卫生—肿瘤]