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 1190 +/- 63, 1 227 +/- 69, 1272 +/- 71 and 1326 +/- 82, respectively. One of the squmous metaplasia tissues was chosen and repeated 2-DE for 3 times. Averagely 1216 +/- 75 spots were detected among the 3 gels of metsplasia tissues and 1082 +/- 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.