人大肠腺瘤和腺癌之间相关差异蛋白的蛋白质组学研究

Proteomics study on differentially-expressed proteins involved in the genesis of human colorectal adenoma and adenocarcinoma

目的应用蛋白质组学的方法,建立人大肠腺癌组织、大肠腺瘤组织、正常大肠黏膜组织的二维电泳图谱,筛选并鉴定三者之间的差异蛋白,发现人大肠癌的潜在生物学标志物。方法收集同时患有大肠腺瘤的大肠腺癌患者10例,术前及术后病理证实均为Dukes'B期。取同一患者术后大肠癌组织、距大肠癌组织15cm以上正常大肠黏膜组织以及肠镜下切除大肠腺瘤组织,提取不同组织总蛋白进行二维电泳,用Image Master 2D Platinum 5.0凝胶图像分析软件对双向电泳图依次进行凝胶点的检测、背景消减和编号。识别不同组织之间的差异蛋白点,对差异蛋白点用胶内酶解后行质谱分析和网上数据库鉴定。结果大肠腺癌组织、大肠腺瘤组织和正常大肠黏膜组织之间存在90个差异蛋白点。对这些差异点进行质谱分析,经数据库鉴定出71个蛋白质。经分析,大肠腺瘤组织与正常大肠黏膜组织相比,17个蛋白表达上调,9个蛋白表达下调;大肠腺癌组织与正常大肠黏膜组织比较,47个蛋白表达上调,12个蛋白表达下调;大肠腺癌组织与大肠腺瘤组织相比,35个蛋白表达上调,8个蛋白表达下调。结论大肠腺癌组织、大肠腺瘤组织和正常大肠黏膜组织之间存在差异蛋白表达,可能与大肠腺癌的发生发展有关;应用蛋白质组学方法筛选人大肠腺癌发生发展相关蛋白是可行的。

Objective To explore differentially expressed proteins among normal human colorectal mucous epithelium,colorectal adenoma,colorectal adenocarcinoma by using two-dimensional gel electrophoresis,mass spectrometry and bioinformatics analysis,to find potential protein markers for diagnosing and treating human colorectal carcinoma.Methods Samples were taken from 10 patients with both colorectal carcinoma and colorectal adenoma.Ten patients were Dukes＇ B colorectal cancer,who confirmed by preoperative and postoperative pathology.The fresh carcinoma tissues were cut off immediately after the specimen resected from the body.The normal mucous tissues at the location far from the tumor over than 15cm were cut off in the same time.The adenoma tissues were cut off under colorectal endoscopy.Protein separation was performed by two-dimensional gel electrophoresis（2-DE）,the images of the gels were acquired by the scanner and then analyzed by Image Master 2D Platinum 5.0 software to find the differentially expressed protein-spots in each tissue.Further,the differentially expressed protein-spots were incised from the gels and digested by trypsin.The peptide mass fingerprinting（PMF） were detected by MALDI-TOF-MS/MS and identified the proteins by searching the Mascot-database.Then we did bioinformatics analysis among the differentially expressed protein-spots.Results All the gels were successfully analyzed by Image Master 2D Platinum 5.0 software;90 differentially expressed protein-spots were found and incised from the gels and digested by trypsin.The PMF of 71 proteins were detected by MALDI-TOF-MS/MS and identified by searching the Mascot-database.Seventeen proteins were upregulated and 9 were downregulated in adenoma compared with the same protein in matched normal mucous tissue.Forty-seven were upregulated,and 12 were downregulated in carcinoma compared with the same protein in matched normal tissue.Thirty-five proteins were upregulated and 8 were downregulated in carcinoma compared with the same protein in matched adenoma tissue.We make a series of functional annotation of the differentially expressed proteins,such as biological process,molecular function and cellular component.We pointed some pathways involved in the genesis of colorectal carcinoma and colorectal adenoma according to the founding of differentially expressed proteins.Conclusion There are some differentially expressed proteins exist among colorectal carcinoma,colorectal adenoma and normal mucous tissue.Proteomics method can be used to seek candidate proteins and pathways in the genesis of colorectal adenoma and colorectal carcinoma.