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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 ,初步建立了分辨率较高
OBJECTIVE: To improve and optimize the preparation method of protein samples in bronchial epithelial tissues and to establish 2 DE patterns of tissues in various stages of human bronchial epithelial carcinogenesis, which will lay the foundation for identification and identification of cancerous related proteins in squamous cell lung cancer. Methods: Human bronchial epithelium, squamous metaplasia, atypical hyperplasia and epithelial invasive carcinoma were collected and screened. The total protein of bronchial epithelial cells was purified by modified deoxycholate trichloroaeticacid (DOC TCA) method. The total proteins of the tissues at different stages were separated by two-dimensional solid-phase pH gradient gel electrophoresis. The gel was stained by silver staining and identified by ImageMaster 2D software analysis of two-dimensional electrophoresis. Results: Two-dimensional gel electrophoresis of the total protein of bronchial epithelial cells purified by modified DOC TCA method could obtain the two-dimensional gel electrophoresis of human bronchial epithelial tissue with high resolution and good reproducibility. The protein spots of the four kinds of tissue gels of normal epithelium, squamous metaplasia, dysplasia and invasive carcinoma were 1 1 90 ± 6 3, 1 2 2 7 ± 6 9, 1 72 ± 71, 1 32 6 ± 82 . The same squamous metaplasia tissues were selected for 3 replicate tests. The average protein spots of the three gels were 1 2 1 6 ± 75, the average matching points were 1 0 82 ± 6 7, the matching rate was 89.3%, and The three gums had good repeatability at the protein spots. The deviation of the protein spots in the IEF direction was (0 .835 ± 0.247) mm and the deviation in SDS PAGE was (0. 92 1 ± 0 .1 0 4) mm. Conclusion: The modified method of DOC TCA precipitation is a good method for the preparation of samples of bronchial epithelial protein. The method of pre-established high resolution