| Epstein-Barr virus (EBV) is a tumor-related virus and EBV genome exists in many human malignant tumors, such as burkitt’s lymphoma, hodgkin’s disease, nasopharyngeal carcinoma and gastric carcinoma. EBV infected cells present a serial changs in micro-molecular metabolism, especially in protein synthsis pathway. These unnormal responses lead a difference between infected cells and non-infected cells in cellular protein expression spectrum. These proteins are the executors which virus infect host cells and host cells resist virus infection. Thus, combining comparative proteomics in viral infection research is a powerful strategy, which can discovery the new clues and find new proteins to clarify the mechanism of biological changes induced by EBV. In this study, we analyze the differential expression proteins in EBV-infected gastric epithelial AGS cells in a comparative proteomic way. Thus, this work provides an important insight into host cell proteome changes during EBV infection and new clues to facilitate further investigation of the underlying mechanism of EBV infection and EBV pathogenesis in gastric carcinoma.This dissertation consists of three parts and the contents are summarized as follows:In the first chapter, a review of the progress of EBV infection and gastric cancer was presented. Then, we summarized the progress of the proteomics and comparative proteomics research in virology. In this study, we combined the comparative proteomics with virology, and proposed the research direction in this work.In the second chapter, we constructed, cultivated and identified of the EBV infection model of gastric epithelial AGS Cells. For EBV infection, EBV-negative human gastric epithelial cell line (AGS) was stably infected in vitro with a recombinant EBV using the cell-to-cell contact method. Based on our previous study, the EBV infection was verified by in situ hybridization and electronic microscope. The result of in situ hybridization showed that EBV EBER-1can be detected nearly100%in the nuclei of the EBV infected AGS cells. The EBV particle could be observed in the EBV infected AGS cells under electronic microscope.In the third chapter, comparative proteomic analyzed the Epstein-Barr virus-infected gastric epithelial AGS cells. To analyse the changes in cell protein profiles between non-and EBV infected host cell, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) was used to analyze the differentially expressed proteins. From the results of2-DE analysis, we found11differential protein spots which expression level changed above two fold. There are9protein spots were successfully identified using the methods of MALDI-TOF and LC-ESI-IT spectrometry mass, including5up-regulated protein spots and4down-regulated protein spots. Our studies revealed that EBV infection induced the increased expression of heat shock cognate71kDa protein, actin cytoplasmic l,pyridoxine-5’-phosphate oxidase, caspase9, t-complex protein1subunit alpha.In addition, EBV infection induced decreased expression of zinc finger protein2, cyclin-dependent kinase2, macrophage-capping protein, growth/differentiation factor11.To test the reliability of the mass spectrometry results, we compared the cyclin-dependent kinase2and caspase9expression between non-and EBV infected cells by western-blot. Results showed that EBV infection suppressed the expression of cyclin-dependent kinase2and increased the expression of caspase9. This results were consistent with our mass spectrometry results.The cyclin-dependent kinase2, which is down-regulated in EBV-infected AGS cells, is one of the most important regulators of the cell cycle, we speculated that EBV alter cell regulation, causing cell cycle disorder, and lead to malignant proliferation of cells. The caspase9, which is up-regulated in EB V-infected AGS cells, is one of the principal biocatalysts in executing cell apoptosis. we speculated that that regulation of apoptosis could be one of the defense mechanism against EBV infection. The down-regulation expression of cyclin-dependent kinase2and up-regulation expression of caspase9in EBV-infected AGS cells might be host cell specific proteins and imply its special roles in EBV infection. Other proteins are waiting for further confirming. Our study provided useful protein-related information to facilitate further investigation of the mechanisms underlying EBV infection and pathogenesis. |
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