结直肠癌化疗敏感性相关蛋白的蛋白质组学初步研究

Proteomic identification of chemosensitivity-associated proteins in human colorectal carcinomas

目的:本研究应用蛋白质组学技术建立化疗敏感性不同的结直肠癌组织总蛋白双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)图谱,并鉴定部分差异表达蛋白,以发现与结直肠癌化疗敏感性有关的蛋白。方法:收集临床诊断为晚期结直肠癌的病例,肠镜活检获取新鲜结直肠癌标本后液氮保存备用,根据肿瘤药物敏感实验分为化疗高敏感组和化疗低敏感组。提取组织总蛋白,采用双向凝胶电泳技术得到各组凝胶图谱;采用PD-quest7.3软件进行图像的合成、对比和差异分析,识别化疗高敏感组和化疗低敏感组之间差异表达的蛋白斑点;选取差异蛋白质点,胶内酶解后行肽指纹图分析及网上数据库检索,鉴定差异蛋白质;应用Western印迹法检测部分差异蛋白的表达情况。结果:建立了化疗高敏感组和化疗低敏感组的双向凝胶电泳图谱,多数蛋白质点集中于pH4～8、相对分子质量为(20～100)×103。高敏感组和低敏感组的电泳图谱中平均蛋白质点数分别为(842±23)个和(793±19)个,2组平均匹配率为90.7%,2组间差异表达蛋白质点数为(79.00±13.56)个;选择30个差异蛋白质点进行质谱分析,经数据库查询鉴定出9个差异表达蛋白。结论:在化疗敏感性不同的结直肠癌中存在蛋白质表达的差异,这些差异表达蛋白可能与化疗敏感性有关,并可能用于化疗敏感性的预测。

Objective：The study aims to screen chemosensitivity-associated proteins in colorectal carcinoma tissues by using two-dimensional gel electrophoresis （2-DE） and mass spectrometry,then identify some differentially-expressed proteins. Methods：The patients with advanced colorectal carcinoma were confirmed by clinical diagnosis. Fresh carcinoma specimens were collected by biopsy and preserved in liquid N2. The tissues were classified into two groups：high sensitivity group （HS） and low sensitivity group （LS） based on drug sensitivity test. The total proteins were extracted and separated by 2-DE. The images were composed,compared,and differentially analyzed to identify the proteins with differential expression in HS and LS groups. Then the differentially-expressed protein spots were incised from the gels and digested by trypsin. The peptide mass fingerprintings （PMF） was acquired after matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS） and the proteins were identified by data searching in the Mascot database. Two proteins with differential expression were detected by Western blotting.Results：The 2-DE spectrum of HS and LS groups were established. Most protein spots were distributed in the area with pH 4-8 and relative molecular weight of （20-100）×103. The average number of the protein spots was 842±23 in HS group and 793±19 in LS group,respectively. The mean matching rate was 90.7%. The number of differentially-expressed dots between HS and LS group was 79.00±13.56. Thirty protein dots were selected for mass spectrum and bioinformatic analysis,and 9 proteins were identified. Conclusion：Colorectal carcinoma with different chemosensitivity had differential protein expression profiles. The differentially expressed proteins may be associated with chemosensitivity and could be used for prediction of chemosensitivity of colorectal carcinoma.