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

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.