| Background and objectives:Nasophryngeal carcinoma(NPC) is one of the common malignant tumors in China, especially in the southern part of the country. Radiotherapy is considered to be a prevalent treatment in present. However, some clinical patients present local recurrence and local residue after radiotherapy, one of the important reasons is due to the radiation resistance in cancer cells. Although there are a lot of research both at home and abroad at the gene level to study the mechanisms of tumor resistance to radiation, which found that the genes such as cell cycle regulation,apoptosis and DNA damage repair are associated with radiation resistance in nasopharyngeal carcinoma, no single gene to be a good predictor for determing the development of tumor radiation resistance and prognosis. It could be presume that the main reason may be the expression of mRNA abundance was inconsistence with its corresponding protein level. From this information, we can learn that the occurrence of nasopharyngeal radioresistance is not caused by a single gene or protein, but the result of a molecular network interaction which is constituted by multiple genes or proteins. At present, the proteomics research of NPC have focused on early diagnosis of nasophryngeal carcinoma and screening of tumor markers and so on. To study the in vitro culture tumor cells but no tissue samples, because it can be avoided the clinical heterogeneity,complex influence factors,the easily lost of positive results and many other shortcomings. Thus, began to study from in vitro cultured tumor cell lines gradually transition to clinical pratice has becomed one of the main way to learn the mechanisms of radiation resistance.The purpose of our study is through the application of proteomics technology to search for radioresistance associated candidate proteins, We first establish and optimize the method for preparation the protein sample , and then the radioresistant human nasopharyngeal carcinoma cell line CNE-2R and its parentall cell line CNE-2 were performed by comparative proteomics, and then discover the differentially expressed proteins which associated with radioresistance. We aim to initially clarified the molecular mechanism of radiation resistance of NPC at the protein expression level, which lay the foundation for the follow-up verification of biology,functional and animal experiments, which also provide a new ideas for the clinical prediction of individual radiosensitivity of the nasopharyngeal carcinoma patients and the implementation of individual radiation therapy in the future. Methods1,Three different ways were adopted to take out the proteins of human nasopharyngeal carcinoma cell line CNE-2, and then the proteins were separated by two-dimensional electrophoresis, establish a better stability and repeatability method for reparation the cell protein sample.2,The total proteins were extracted from radioresistant human nasopharyngeal carcinoma cell line CNE-2R and its parentall cell line CNE-2 and were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), which was respectively run by Isoelectric Focusing Electrophoresis (IEF) as the first dimensional electrophresis, and then horizontal SDS-PAGE as as the first dimensional electrophresis.3,After electrophoresis, stained with silver nitrate, ImageMaster 2-DE Platinum 5.0 software was used to analysis gels, differentially proteins which average spot intensity was showed a difference >2-fold were cut from gels, and then were bleached, hydrolysised, extracted. Which lay the foundation for subsequent mass spectrometry analysis.4,The differentially expressed proteins were identified by Matrix-assisted laser desorption/ionization-time-of-flight tandem Mass Spectrometry (MALDI-TOF-MS) technique, and searching in the Mascot protein database for protein identification. With the help of artificial literature database search, the functional protein could be futher reserch.Results1,The combination of the General lysate and Destreak reagent can get a 2-DE map with better image clarity and higher resolution.2,The total proteins were extracted from radioresistant human nasopharyngeal carcinoma cell line CNE-2R and its parentall cell line CNE-2 and were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), then high quality 2-DE profiles were obtained and analyzed by ImageMaster 5.0 software to screen differentially expressed protein spots,there were 32 protein spots which average spot intensity was showed a difference >2-fold.3,Those 32 spots were identified by matrix-assisted laser desorption/ioniation time-of-flight tandem mass spectrometry. of which 11 protein spots were successfully identified, Bioinformatics analysis showed that most of the proteins were signal transduction related proteins,molecular chaperone,cytoskeletal components, which were mainly associated with the biological function such as apoptosis regulation,cell cycle regulation,RNA transcription,signal transduction,cytoskeletal components and radiation stress and so on. These proteins can be used as a potential target for radiation therapy, as an effective way to improve tumor radiosensitivity, and provide a new idea and methods for nasopharyngeal cancer radiation therapy research.Conclusion2-DE combined with MALDI-TOF-MS technology can be effectively recognized and identificated the differentially expressed proteins of radioresistant human nasopharyngeal carcinoma cell line and its parentall cell line. Our study found 11 differentially expressed proteins, their functions involved in many physiological metabolism and adjustment process, which suggesting that the radiation resistance of nasopharyngeal carcinoma may be the result of the interaction of multiple proteins , which may be involved in the occurrence of it。... |
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