目的 运用蛋白质组学经典技术,双向凝胶电泳、质谱鉴定及生物信息学分析在胆囊癌组、正常胆囊对照组中寻找差异蛋白,发现和鉴定胆囊癌相关的肿瘤标志物及潜在的治疗靶点。方法 标本分两组:胆囊癌组-胆囊癌组标本59例;正常对照组-胆囊癌癌旁组织标本59例。组织样品通过洗涤、裂解、离心收集总蛋白样本,分装冻存。经过第一向等点聚焦、第二向SDS-PAGE电泳,凝胶经考马斯亮蓝染色显色后,用Gs-800光密度仪获得凝胶图像。经PD-Quest凝胶图像分析软件进行分析后,找出有差异的蛋白点。通过蛋白指纹谱鉴定(PMF), 基质辅助激光解吸附质谱技术(MALDI-TOF) 获得差异蛋白碎片的二级质谱,利用Biotools软件采集肽碎片质荷比信息;利用Mascot搜索引擎对数据库进行检索,鉴定差异蛋白质的氨基酸序列,获得相应特异蛋白质信息。结果 发现66个差异表达蛋白。其中在胆囊癌中上调表达蛋白21个,下调表达蛋白45个。21个差异蛋白得分大于56,在胆囊癌中上调表达的10个,下调表达的11个。主要包括膜联蛋白、膜突蛋白、热休克蛋白、波形蛋白、视黄醛脱氢酶 1、半乳凝素等。结论 上述蛋白可能在肿瘤的发生发展机制中起着重要的作用,是诊断、治疗的潜在靶点,值得后续进一步研究。
Abstract
Objective To identify the differential proteins in gallbladder cancer group and normal gallbladder tissue control group, and find and identify the tumor markers related to gallbladder cancer and potential therapeutic targets by using classical proteomic techniques, two-dimensional gel electrophoresis, mass spectrometry identification and bioinformatics analysis. Methods The samples were divided into two groups: gallbladder cancer group (59 cases) and normal control group (40 cases). The total protein samples were collected by washing, lysis and centrifugation of the tissue samples, and then packaged and frozen. After the first direction isofocal focusing and the second SDS-PAGE electrophoresis, the gel was dyed and colored with Coomnassie blue, and the gel image was obtained by Gs-800 optical densitometer. After analysis by PD Quest gel image analysis software, different protein spots were found. Secondary mass spectrometry of differential protein fragments was obtained using Peptide Mass Fingerprinting (PMF) and Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry techniques. Biotools software was used to collect mass- charge ratio information of peptide fragments. The Mascot search engine was used to search the database, identify the amino acid sequences of differential proteins, and obtain the corresponding specific protein information. Results A total of 66 differentially expressed proteins were found, Among which, 21 proteins were up-regulated and 45 proteins were down-regulated. Of 21 differential proteins with scores higher than 56, 10 were up-regulated and 11 were down-regulated in gallbladder cancer. It mainly includes annexinsANX, moesin, heat shock protein (HSP), vimentin, Retinol dehydrogenase 1, galectin, and so on. Conclusions The above-mentioned proteins may play an important role in the occurrence and development mechanism of tumors, and are potential targets for diagnosis and treatment, which deserves further research in the future.
关键词
胆囊癌 /
组织蛋白质 /
双向凝胶电泳 /
质谱分析
Key words
gallbladder cancer /
tissue proteins /
two dimensional gel electrophoresis /
mass spectrometry
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