| Oral submucous fibrosis (OSF) is a kind of chronic and complicated oral mucousal disease. It is characterized by juxta-epithelial inflammatory reaction and progressive fibrosis of the submucosal tissues. The main aetiological factor of OSF is betel quid chewing. OSF is a highly potent pre-cancerous condition which has been regarded as oral pre-cancerous lesion. The malignant transformation rate of OSF was7%～13%. The researchers have paid more attention to the pathogenesis and malignant transformation of OSF. Our group has studied the gene expression profiles of OSF and normal buccal mucosa (NBM) tissues by using the Affymetrix U133A2.0chips, analyzed the differentially expressed genes by using bioinformatics tools, and further validated several genes. The genomics study could not reveal the whole molecular mechanisms of pathogenesis of OSF. To find more new molecular biomarkers and therapeutic targets of OSF, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MADLDI-TOF-MS) was performed to screen and identify the differential proteins in the buccal mucosa of OSF. The differential expression levels of five differential proteins were further validated by Western blotting and immunohistochemistry.1. Proteomic screening and identification of differential expressed proteins in OSFObjective:To screen and analyze differential expressed proteins of OSF.Methods:We obtained10NBM tissue samples and10OSF buccal mucosa tissue samples, which were then pooled into one pair of samples. The proteins extracted from the tissues were separated by2-DE, respectively. The gel images were analyzed by software and the differential protein spots were then analyzed by mass spectrometry. The identified proteins were annotated and Gene ontology mapped.Results:A total of48differential protein spots, whose fold change value were more than2, were obtained by2-DE. At last,33differential expressed proteins between NBM and OSF were identified after mass spectrometry analysis and database searching. Among these proteins,22proteins were up-regulated in OSF and11proteins were down-regulated.Conclusion:The differential expressed proteins in OSF were efficiently and accurately screened and identified in our comparative proteomic study. These findings could provide us with some new data in searching the useful specific molecular biomarkers in the pathogenesis of OSF.2. Validation of partial differential expressed proteins in OSF by Western blotting and immunohistochemistry.Objective:To validate5differential expressed proteins of OSF and investigate their clinical significances.Methods:Twenty NBM with20OSF samples and20NBM with60OSF samples were obtained for Western blotting and immunohistochemistry, respectively. The semi-quantitative and qualitative expression analysis of5differential proteins (prohibitin, peroxiredonxin2, translationally controlled tumor protein, calumenin and cellular retinoic acid binding protein1) in NBM and OSF were performed.Results:Western blotting analysis showed that the expressions of prohibitin, peroxiredonxin2and translationally controlled tumor protein were significantly up-regulated in OSF, and the expressions of calumenin and cellular retinoic acid binding protein1were significantly down-regulated in OSF. The results of immunohistochemistry showed that the positive expression rates of prohibitin, peroxiredonxin2, translationally controlled tumor protein, calumenin and cellular retinoic acid binding protein1were70.0%,71.7%,81.7%,8.3%and0%, respectively. The former three proteins were significantly over-expressed and the latter two proteins were significantly down-expressed in OSF samples than in NBM samples, respectively.Conclusion:The results of Western blotting and immuno-histochemistry coincided with the data of the comparative proteomic analysis. The differential expressions of three up-regulated and two down-regulated proteins may affect some biological behavior of OSF, explain some pathobiological processes, and could be potential molecular biomarkers for OSF pathogenesis.
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