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.