【摘要】 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更多还原

【Abstract】 BACKGROUND &OBJECTIVE: The carcinogenesis of bronchial epithelial cells is a complex multiple stage process involving multiple genes, but its mechanism remains unclear. Studying this process with proteomic approaches may identify carcinogenesis associated proteins, which are important for elucidating carcinogenic mechanism of human lung squamous carcinoma. This study was designed to optimize the protein preparation methods for bronchial epithelial tissues, to establish two dimensional gel electrophoresis profiles of human bronchial epithelial tissues from different stages in carcinogenic process, and to perform differential analysis and provide a basis for identifying carcinogenesis associated proteins of lung squamous carcinoma. METHODS: After obtaining samples of the normal, metaplasia, dysplasia, carcinoma tissues of human bronchial epithelia, modified deoxycholate trichloroaetic acid (DOC TCA) precipitation was used to extract and purify the total proteins of bronchial epithelial samples. Immobilized pH gradient two dimensional polyacrylamide gel electrophoresis (2 DE) was used to separate the total proteins of the samples. After silver staining, ImageMaster 2 DE image analysis software was applied to analyze 2 DE images. Some selected differential protein spots were identified by peptide mass fingerprint (PMF) based on matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) and database search. RESULTS: The total proteins extracted with the method described here were used to perform 2 DE. 2 DE patterns with high resolution and reproducibility from different stages were obtained. The average spots for normal epithelium, metaplasia, dysplasia and invasive carcinoma were 1189.50±39.89, 1227.00±37.90, 1273.00±43.31, and 1326.00±66.63, respectively. The test was repeated, which showed that there were average 1216 ±75 spots among 3 gels of the same metaplasia tissues and 1082 ±67 spots were matched. The average matching rate was 89.3%and protein spots in 3 gels had good reproducibility. The average position deviation of matched spots in different gels was 0.865±0.247 mm in IEF direction, and 0.971±0.104 mm in SDS PAGE direction. The 2D images of 32 samples were compared, which showed a significant difference of the number of average protein spots among the four groups (P< 0.05). The average differential spots between the low and advanced stages were 31.50 ±7.67, 41.00 ±9.07, and 56.00 ±8.96, respectively. Twenty three protein spots chosen randomly from dysplasia and invasive carcinoma groups were identified by PMF, some of which were involved in the cell proliferation, differentiation, cycle regulation, signal transduction and tumor occurrence. CONCLUSIONS:(1) Improved DOC TCA precipitation is a preferable method for protein preparation from bronchial epithelial tissues. (2) We established well resolved, reproducible 2 DE profiles of human bronchial epithelial tissue in different stages of carcinogenesis. Some differentially expressed proteins may be related to carcinogenesis of bronchial epithelia. These results provide a fundamental basis for further study of carcinogenic mechanism of lung squamous cancer and screening its specific marker.更多还原