大肠癌临床分期相关蛋白的蛋白质组学研究

目的：筛选与大肠癌临床分期相关的蛋白，为大肠癌分子分期和预后预测提供依据。方法：将不同临床分期大肠癌组织蛋白进行二维凝胶电泳，选择部分差异表达蛋白进行MALDI-TOF质谱分析和生物信息学分析以鉴定差异表达蛋白;免疫组织化学方法验证筛选结果。结果： 建立了不同临床分期大肠癌组织的二维凝胶电泳图谱，其中Ⅰ，Ⅱ，Ⅲ，Ⅳ大肠癌组织平均蛋白质点数分别为970±41,980±32,1010±43,1240±34;以Ⅰ期大肠癌为参照，Ⅱ期大肠癌差异表达蛋白有52.00±12，Ⅲ期大肠癌差异表达蛋白42.00±11，Ⅳ期大肠癌差异表达蛋白72.00±15，通过进行质谱分析和生物信息学查询，鉴定30个显著差异表达的蛋白点，其中Ⅱ，Ⅲ，Ⅳ均上调的有3种蛋白：AnnexinⅡ,AnnexinⅣ,热休克蛋白27(HSP27)。而仅在Ⅳ期中上调蛋白有1种蛋白,即肝脂肪酸结合蛋白（LFABP)。AnnexinⅡ和肝型脂肪酸结合蛋白表达的免疫组化检测结果与蛋白质筛选结果基本一致。结论：不同临床分期的大肠癌中存在着差异表达蛋白，这些蛋白可能作为大肠癌分子分期和预后的标志物。

Proteomic Study of associated proteins of clinical stage of colorectal carcinoma

Abstract:Objective：To screen the associated proteins of clinical stage of colorectal carcinoma(CRC) in order to provide the basis of molecular biological stages and outcome prediction of CRC. Methods ：Total protein from colorectal carcinoma tissues were extracted, differential proteome profiles were established and analysized by means of immobilized pH gradient-based two-dimesional polyacrylamide gel electrophoresis(2D-PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Immunohistochemical method was used to assess the results mentioned above. Results：Well-resolved, reproducible 2-DE profiles of human colorectal carcinoma tissues were obtained. Avarage protein dots were 970±41，980±32，1010±43，1240±34 in stageⅠ, stageⅡ, stageⅢ, stageⅣ, respectively; compared to stageⅠ, differential expressed protein dots was 52.00±12 in stageⅡ; 42.00±11 in stageⅢ; 72.00±15 in stageⅣ ;30 differentail expressing proteins were analysized by mass spectrometry and bioinformation, part of them were well characterized . Three proteins (Annexin Ⅱ,Annexin Ⅳ and HSP27) were overexpressed in stageⅡ, stageⅢ, stageⅣ, and Liver fatty acid-binding protein(LFABP) was only overexpressed in stageⅣ. Resullts of immunohistochemical assay for AnnexinⅡ and LFABP were consistant with proteomic results. Conclusions：Differentail expressed proteins exist in different clinical stage of CRC, which would be as biomarkers for diagnosis and prediction of prognosis of CRC.