Proteomics Of Exosome Derived From U251 Glioma Cell And Glioma

Glioma is the most common malignant tumor of the nervous system. It is a originated from neuroepithelial cell. At present the main treatment for glioma is surgical treatment supplemented with a comprehensive treatment of radiotherapy ,chemotherapy and immunotherapy. Due to the high-invaded and high recurrence rate, high mortality,all these therapies have done little to improve the poor prognosis rate for patients with these dreaded tumors。With the development of molecular biology and tumor immunology,people are becoming aware that the Glioma immune therapy is very promising.Their researchs include Cell vaccines, molecular vaccines and immune gene therapy and so on. Although they have achieved some success in pre-clinical studies,but there is still a long way for immunotherapy being clinical anti-cancer therapy against gliomas.1987, Rose Johnstone found some vesicles secreted by Reticulocyte and he termed them exosome.After that,more and more reseachers have done the related researches,they found lots of cells such as blood cell, epithelial cell, neurocyte, tumor cell(include melanoma cell,lung cancer cell, celiothelioma cell,breast cancer cell,ovarian cancer cell, leukemic cell and so on) can secret exsome.Some researchers found exosomes contain MHC class II and class I molecules, heat shock proteins, costimulatory molecules (such as CD86 andICAM-1) and so on. Exosomes derived from tumor cells contain the tumor-associated antigen. Except carrying abandoned protein from the cells,exsomes can transmitt MHCⅠ,Ⅱelements to the APC cells, activated T cells, then expanded the effect of immune response.Some researcheres found DC derived exsome showing a different level of anti-tumor effect in vivo and without any toxicity in clinical trials so far.Due to the anti-tumor immune response of tumor cell derived exosomes, we open up a new anti-tumor therapeutic approaches .However, proteomics research of glioma cell derived exsome has not been reported.our research will define glioma cells in vivo or in vitro can excrete exosomes.Then we will take a proteomics research of glioma cell derived exosomes.Our research will provid the basic theory for anti-glioma response of exosomes and lay the foundation for further study.Methods:1,Extracting exosome from hydatid fluid of malignant glioma and culture fluid of U251 by gradient centrifugation, observing the morphology of glioma cell derived exosome by electron microscope2,Extracting proteins in samples and determinng the protein content of samples by Bradford method. seperating the protein by 2D-PAGE and analysing the results by software imagemaster2Dplatinum.3, By probing the protein database swissprot and TrEMBL with keyword exosome and searching the pI and MW of these proteins in the schedule we will obtain the preliminary proteome information of glioma cell derived exosome 4,Identification the protein component of glioma cells derived exosome by mass spectrogramResults:1, Observing samples by electron microscope ,we find some round or oval-shaped vesicles with the diameter between 60-150nm on average of 100nm..2, After analyzing the results of 2D-PAGE,we got 270 spots from the sample of U251 and 66 spots is consistent with the database.We got 242spots from the sample of glioma's hydatid fluid malignant and 60 spots is consistent with the database. We find glioma cell dereived exosome contains RNA-binding protein, exonuclease, HSP70, MHC class I, MHC class II and probably contains Cancer-Testis Antigen, vascular endothelial growth factor and so on..3, MS analysis identified a total of 17 proteins.They are low density lipoprotein receptor, E1-E2 ATPase,TCR chainβ, eukaryotic translation initiation factor 3, heavy and light chain of immunoglobulin,crystal structure of the Igg1 FC-fragment, heavy chain of B cell antibody, VKORC1,chromosome 1 open reading frame 142, thyroid hormone stabilize factor,hCG, nucleotide exchange factor,vitamin-D binding protein precursor,glyoxalase domain containing protein 4, some hypothetical protein and unnamed protein.Conclusion:1,Glioma cells artificial breeded or in vivo can excrete exosome.2,All the data obtained from our research and other reports have reveals a common constituent between glioma cell derived exsome and other cells.3,We find glioma cell dereived exosome contains RNA-binding protein, exonuclease, HSP70, MHC class I, MHC class II,CT antigen and VEGF.This reveals some properties of immunomodulation for glioma derived exosome.The novel points:1,We first find the glioma cell derived exosome2,The proteomic analysis of glioma cell derived exosome is initially performed...