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背景与目的:支气管上皮细胞的癌变是一个多基因参与、多阶段的复杂过程,但癌变机理仍不清楚,应用蛋白质组学技术研究此过程有可能识别癌变相关蛋白质,对揭示肺鳞癌癌变机制具有重要的意义。本研究的目的是优化支气管上皮组织的蛋白质样品制备方法,建立人支气管上皮癌变各阶段组织的2-DE图谱并进行差异分析,为鉴定肺鳞癌癌变相关蛋白质奠定基础。方法:收集、筛选人支气管正常上皮、鳞状上皮化生、不典型增生和上皮浸润癌组织标本。改良的脱氧胆酸-三氯醋酸(deoxycholate-trichloroaeticacid,DOC-TCA)法提纯支气管上皮总蛋白质,应用固相pH梯度双向凝胶电泳分离各阶段组织的总蛋白质,凝胶经银染显色后,用ImageMaster2D软件分析图谱,比较差异表达蛋白质。应用基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtime-of-flightmassspectrometry,MALDI-TOF-MS)得到相应的肽质指纹图谱(peptidemassfingerprint,PMF),然后搜索数据库鉴定部分差异蛋白质。结果:应用改良的DOC-TCA法提纯总蛋白质进行双向电泳,获得了分辨率高、重复性好的人支气管上皮组织的双向电泳图谱,支气管正常上皮、鳞状上皮化生、不典型增生和上皮浸润癌组织凝胶的平均蛋白质点数依次为1189.50±39.89、1227.00±3
BACKGROUND & OBJECTIVE: The carcinogenesis of bronchial epithelial cells is a multi-gene involved, multi-stage complex process. However, the mechanism of carcinogenesis remains unclear. Using proteomics to study this process may identify cancer-associated proteins. It is of great significance. The purpose of this study is to optimize the preparation of protein samples of bronchial epithelial tissue and to establish the 2-DE map of the different stages of human bronchial epithelial carcinogenesis and to carry out the differential analysis, which will lay the foundation for the identification of cancer-related proteins in squamous cell lung cancer. Methods: Human bronchial epithelial cells, squamous metaplasia, dysplasia and epithelial infiltrating carcinoma tissues were collected and screened. The total protein of bronchial epithelial cells was purified by modified deoxycholate-trichloroethacic acid (DOC-TCA) method. The total proteins of the tissues at different stages were separated by two-dimensional solid-phase pH gradient gel electrophoresis. , Using ImageMaster2D software analysis of the map, the more differentially expressed protein. The corresponding peptide mass fingerprint (PMF) was obtained by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and then the database was searched to identify partial differential proteins. Results: Two-dimensional gel electrophoresis (SDS-PAGE) was used to purify total protein with modified DOC-TCA method. The two-dimensional gel electrophoresis pattern of human bronchial epithelial tissue with high resolution and good reproducibility was obtained. The normal bronchial epithelium, squamous metaplasia, dysplasia and epithelium The average number of protein spots in invasive carcinoma tissues was 1189.50 ± 39.89 and 1227.00 ± 3