Proteomic Analysis Of Human Gastric Carcinoma Cells With Different Levels Of COX-2 By Two-Dimensional Electrophoresis

COX-2 is highly expressed in most of gastric cancer cell lines and tissues. COX-2 activity inhibition can lead to low proliferation and high apoptosis rate of gastric cancer cells. All of these indicated that COX-2 plays an important role in development of gastric cancer. However, the mechanisms is not clear. Proteomics methods can be used to compare differentiated-expressed protein profiling of different cells and samples. Our work was focused on construction and optimization of two-dimensional gel electrophoresis method and analyzing protein profiling of gastric cancer cells SGC7901-asCOX2 and SGC7901-pcDNA, which were respectively constructed by transfecting SGC7901 with antisense and empty vector of COX-2. The differentiated-expressed proteins was visualized, cut, trypsinized and analyzed by MALDI-TOF. The main methods and results were shown as following:1. The total proteins of gastric cancer cells SGC7901-asCOX2 and SGC7901-pcDNA were originally seperated by using the technique ofimmobilized gradient isoelectric focusing gel ( first-dimensionalelectrophoresis, IEF) . The proteins with different pI were seperated horizontally according to the electric charge.2 , The second-dimensional separation removed from equilibration solution was performed on sodium dodecyl sulfate(SDS)-polyacrylamide gels.The proteins with different molecular weight were separated vertically by SDS-PAGE.3, Following SDS-PAGE, brown protein spots or strips were visualised using Silver Staining method. The proteins were combined with Ag+ and the silver was separated after the deoxidization with formaldehyde.4, ImageMaster image software and Melanie3 software was respectively, used to seize and analyze the photoes of two dimensional gels. Our results showed that 6 protein spots can only be detected in SGC7901-pcDNA and 4 spots in SGC7901-asCOX2. Compared with SGC7901-pcDNA, the expression of 10 protein spots was downregulated and 6 spots was upregulated.5 , The spots were excised and destained, then they were deoxidized and alkylated and at last they were trypsinized and extracted. Finally the special peptide segment was obtained.6, MALDI-TOF MS was performed after emendation and the data of peptide mass fingerprinting was obtained.7, Peptide matching and protein searches were performed automatically with the use of data-base software ( Mascot ) and the website ( Http://www.matrixscience.com) .All these results suggested that the difference between the proteomes of...