The Proteomics Study Of Altered Gene Expression In CNE2 Cells Treated With EBV And TPA Respectively And Cooperatively

Nasopharyngeal carcinoma(NPC) is an endemic cancer with a high incidence in Southeast Asia and Southern China, with obvious inclination towards racial and geographic distribution. Former studies indicate that EB virus, genetic factors, dietary, and certain environmental factors are considered to be associated with this cancer. Although the mechanism involved in the initiation and promotion of NPC is unclear, more and more data are in favor of that EBV is the primary pathogen for this cancer, and then the role of EBV in NPC is increasingly emphasized.To clarify the mechanism of NPC development, we should solve the key problem how the EBV induces the biological changes of nasopharyngeal epithelial cells after infection, rather than emphasize the close relationship between EBV and NPC. However, we have not established an effective model of EBV infecting epithelial cells for a long time, because most epithelial cells, which are absent of classical receptor for EBV－CR2/CD21, are difficult to be infected by EBV. Although some researchers make certain that epithelial cells can be infected by EBV after transfecting CR2/CD21 to them, this artificial manipulation is not enough to explain the mechanism of EBV spontaneous infection and then biological changes induced in cells. Therefore, for clarifying the mechanism of NPC development, it is veryimportant to establish a method by which EBV can effectively enter epithelial cells mimicing the natural condition.In addition, that certain environmental factors like phorbol ester-TPA having profounding influences on EBV infection and NPC incidences also deserves much attention. Researchers are very interested in the mechanism by which TPA induces biological changes of epithelial cells in view of the facts that the geographical distribution of NPC is related to those plants containing TPA and that TPA can make epithelial cells susceptible to EBV infection then be transformed. Therefore, it is worthy of elucidating the difference of expression in molecular level before and after TPA treatment.In our project, we treated nasopharyngeal epithelial cells with EBV and TPA respectively and cooperatively, then analyzed the difference of gene expression in the cells before and after treatment, and found the protein molecules associated with biological changes, to explain the mechanism by which EBV and/or TPA affected epithelial cells. This project took poorly differentiated squamous cell carcinoma cell line-CNE2 of NPCs as experimental target instead of normal primary human nasopharyngeal epithelial cells, because the latter are difficult to be cultivated and cannot proliferate to enough amount for the experiment, moreover, some findings indicated that EBV cannot infect untransformed nasopharyngeal squamous epithelial cells. Referring to the recent studies, this experiment applied a new method-cell-to-cell contact to facilitate EBV infection of epithelial cells. The CNE2 cells were allocated into 4 groups, i.e., untreated control, infected with EBV by cell-to-cell contact, TPA treatment and combining EBV infection and TPA treatment. All the cells were cultivated and treated in the same phase, and then collected to successive experiments.The experiment indicates that cell-to-cell contact is a new feasible method to establish EBV spontaneous infection mode in epithelial cells under the mimic natural condition, since it showed that the B95-8 virus could effectively infect epithelial cells by cell-to-cell contact, which was consistent with former studies on other virus line of EBV.To elucidate the molecular mechanism of biological changes in CNE2 cells induced by EBV and/or TPA, a new technology?functional proteomics" was applied as a study method to identify the genes altered in expression.According to this method, the total proteins from the CNE2 cells of those 4 groups above-mentioned were extracted after lysis and then separated by 2-D electrophoresis respectively, then the protein expression patterns of those 4 group cells were analyzed with certain softwares to f...