人支气管上皮蛋白质样品制备方法及其癌变各阶段组织2-DE图谱的建立

Improvement of protein preparation methods for bronchial epithelial tissues and establishment of 2-DE profiles from carcinogenic process of human bronchial epithelial tissues

目的 :改良、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过程各阶段组织的2 DE图谱 ,为识别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ImageMaster 2D软件分析双向电泳图谱。结果 :应用改良的DOC TCA法提纯的支气管上皮总蛋白质进行双向电泳 ,可获得分辨率高、重复性好的人支气管上皮组织的双向电泳图谱。正常上皮、鳞状化生、不典型增生和浸润癌 4种组织凝胶的蛋白质点数依次为 1 1 90± 6 3,1 2 2 7± 6 9,1 2 72± 71 ,1 32 6± 82。选取同例鳞状上皮化生组织进行 3次重复性检测 ,3块凝胶的平均蛋白质点数为 1 2 1 6± 75 ,平均匹配点数为 1 0 82± 6 7,匹配率达 89.3% ,且 3块胶在蛋白质点位置上有较好的重复性 ,不同胶间蛋白质点在IEF方向的偏差为 (0 .835± 0 .2 4 7)mm ,在SDS PAGE方向上的偏差为 (0 .92 1± 0 .1 0 4 )mm。结论 :改良的DOC TCA沉淀法是一种较好的支气管上皮组织蛋白质样品制备法 。

Objective To optimize the protein preparation methods for bronchial epithelial tissues,to establish two dimensional gel electrophoresis profiles from different stages tissues of human bronchial epithelial carcinogenic process,and to provide a base for identifying carcinogenesis associated proteins of lung squamous carcinoma. Methods After obtaining samples of the normal metaplasia dysplasia carcinoma tissues of human bronchial epithelia, improved 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 seperate the total proteins of the samples .After silver staining, ImageMaster 2 DE image analysis software was applied to analyze 2 DE images . Results The total proteins extracted with improved DOC TCA precipitation was 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 1 190±63, 1 227 ±69,1 272±71 and 1 326±82, respectively. One of the squmous metaplasia tissues was chosen and repeated 2 DE for 3 times .Averagely 1 216±75 spots were detected among the 3 gels of metsplasia tissues and 1 082±67 spots were matched. The average matching rate was 89.3% and protein spots in the 3 gels had a good reproducibility.The average position deviation of matched spots in different gels was 0.835 ±0.247 mm in IEF direction, and 0.921±0.104 mm in SDS PAGE direction. Conclusion Improved DOC TCA precipitation is a proper method for protein sample preparation of bronchial epithelial tissues. The well resolved, reproducible 2 DE profiles from different stage tissues of human bronchial epithelial carcinogenic process have been primarily established.