激光捕获显微切割纯化的人结肠癌组织的定量蛋白质组学研究  

Quantitative Proteomic Study for Human Colon Cancer Tissue by Laser Capture Microdissection

作　　者：李国庆[1,2] 李园园[1,3] 李茂玉[1] 梁旭俊 肖哲锋[1] 邵美英[1] 彭芳[1] 张鹏飞[1] 陈永恒[1] 陈主初[1] 

机构地区：[1]中南大学湘雅医院,卫生部肿瘤蛋白质组学重点实验室,抗癌药物国家与地方联合工程实验室,结构生物学与药物设计湖南省工程实验室,长沙410008 [2]南华大学药学与生物科学学院,湖南省分子靶标新药研究协同创新中心,衡阳421001 [3]广西科技大学医学院,柳州545005

出　　处：《基因组学与应用生物学》2017年第8期3358-3367,共10页Genomics and Applied Biology

基　　金：国家重点基础研究发展计划(973计划)(2011CB910704、2014CBA02004);国家自然科学基金(81272971、81202129、81372516);湖南省教育厅资助科研项目(16C1409)共同资助

摘　　要：为分析结肠上皮癌变进程中的差异表达蛋白质,筛选结肠癌诊断候选标志物,先采用激光捕获显微切割技术(LCM)纯化人正常结肠组织和结肠癌组织,再采用同位素标记对蛋白质进行相对和绝对定量(i TRAQ),然后结合强阳离子柱分离和反相液质联用(2D-LC-MS/MS)鉴定差异表达蛋白质。试验共鉴定了1 042个非冗余蛋白,筛选出与人结肠癌变相关的差异蛋白162个,其中在结肠癌中表达上调的蛋白质68个,表达下调的蛋白质94个,功能分析表明,这些差异蛋白质涉及内质网蛋白质加工、糖酵解/糖原异生、黏着斑细胞连接、调节微丝组装和细胞外基质受体相互作用等分子功能。进一步采用Western blotting及免疫组织化学技术(IHC)验证了其中差异蛋白S100A9的表达,证实了定量蛋白质组学结果的可靠性。研究表明:差异表达蛋白质与结肠癌变相关,并有可能成为结肠癌诊断的候选分子标志物,为进一步深入研究这些蛋白质在结肠癌发生和发展进程中的分子机制,为结肠癌的发病机制研究提供新线索。In order to analyze differentially expressed proteins in carcinogenesis of colonic epithelium and screen candidate markers for colon cancer diagnosis, human normal colon and colon cancer tissues were purified by laser capture microdissection （LCM）. Proteins were quantified by isobaric tags for relative and absolute quantitation （iTRAQ）, then the differentially expressed proteins were identified by strong cation column separation and reverse phase liquid chromatography （2D-LC-MS/MS）. In the research, 1 042 non-redundant proteins were identified and 162 differentially expressed proteins associated with human colon cancer were selected, among which 68 proteins were up-regulated and 94 were down-regulated. The functional analysis showed that these differentially expressed proteins were associated with protein processing in endoplasmic reticulum, glycolysis or gluconeogenesis, focal adhesion junctions, regulation of actin cytoskeleton, ECM-receptor interaction, etc. Western blotting and immunohistochemistry （IHC） were performed to detect the expression levels of S100A9, which also confirmed the reliability of quantitative proteomics results. The study suggested that these differentially expressed proteins were associated with colonic epithelial carcinogenesis, and they might be novel potential biomarkers for detection of colon cancer for further studying the molecular mechanism of these proteins in the development and progression of colon cancer, and it would provide new clues for the pathogenesis of colon cancer.

关 键 词：结肠癌 生物标志物 激光捕获显微切割技术 同位素标记相对和绝对定量 

分 类 号：R735.35[医药卫生—肿瘤]