The Proteomics Study Of Glioma Cell Derived Exosome

Glioma is the most common malignant tumor of the nervous system.At present the main treatment for glioma is surgical treatment supplemented with a comprehensive treatment of radiotherapy and chemotherapy.Due to the high recurrence rate, high morbidity and high mortality,glioma following stroke become the second largest threat to human health of the nervous system diseases.Exosomes are 30-100nm membrane vesicles that are secreted into the extracellular milieu as a consequence of multivesicular body fusion with the plasma membrane.The term "exosome" was first Descripted by Rose Johnstone to vesicles secreted by Reticulocyte in 1987.His opinion was mature red blood cell discarding unnecessary membrane components through exosome in in the final stages of Reticulocyte's maturity.At the same time others found exosome also includes some useful proteins.In 1996,Raposo found B lymphocytes can secrete exosome,Subsequently various tumor cells were confirmed of secreting exosomes.Based on the density and diameter of exosome,application of ultrafiltration membrane and ultracentrifugation method become the conventional means of extracting.We use the method for isolation of ultracentrifugation in our research.Stoorvogel's research of dendritic cells and inflamed human epithelial cells derived exosome reveals a basic component exosome.Exosomes from these cells contain MHC classâ…¡and class â… molecules,cytosolic chaperone proteins,subunits of trimeric G proteins,cytoskeletal proteins,annexins,integrins,enzymes and elongation factors.Savina's research indicated that transferrin(Tf) stimulated exosome release in a Ca²⁺-dependent manner,suggesting that Tf might be a physiological stimulus for exosome release in K562 cells.Wolfers's research about mouse breast cancer cells in vitro revealed exosome contain the antigens of corresponding tumor cells,they also found exosomes can transfer tumor antigens to dendritic cells and induce T-cell-dependent antitumor immunity.In 2005,Escudier produced the vaccine based on the DC derived exosome.Their exosome vaccines Phase I trail was feasible and safe. In the same year,Morse's phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer proved production of the dexosome vaccine was feasible and dexosome therapy was well tolerated in patients with advanced nonsmall-cell lung cancer.Tthere are lots of reserchs on the malignant tumor derived exosome abroad,but specific to the study of glioma-derived exosome are rare.We will extract glioma cell derived exosome and take the proteomics study of glioma cell derived exosome.Objective:To study the protein composition of glioma cell derived exosome.Methods:1.Culturing human glioma cell line SHG44.2.Extracting exosome from hydatid fluid of malignant glioma(sample A)and culture fluid of SHG44(sample B)by gradient centrifugation.3.Observing the morphology of glioma cell derived exosome by electron microscope.4.Extracting proteins in sample A and B by Bradford method.5.the protein was seperated by 2D-PAGE.6.Analysing the results by software imagemaster2Dplatinum after the sivler staining.7.Obtaining the preliminary proteome information of glioma cell derived exosome by probing the protein database.Results:1.Observing samples by electron microscope,we find glioma cells can excrete exosome.2.Using bradford method,we get the protein content of samples A and B were 2.5μg/μl and 2.0μg/μl.3.The samples are separated by 2D-PAGE and then analyzed after silver staining.The results of sample B is not ideal for some unknow reasons.We got 242 spots from sample A and 42 spots from sample B.4.By probing the swissprot database and conferring with the result of sample A,we find glioma cell dereived exosome contains RNA-binding protein,exonuclease,HSP70,MHC classâ… ,MHC classâ…¡and some tumor antigens.Conclusion:1.We find glioma cells can excrete exosome. 2.We find glioma cell dereived exosome contains RNA-binding protein,exonuclease,HSP70,MHC classâ… ,MHC classâ…¡and some tumor antigens.