The Study Of Separation And Purification On Glioma-Derived Exosome

Glioma, a kind of malignant tumor in central nervous system, has a high disease rate which is above 40 percent of all kinds of nervous system tumor. To malignant glioma, we presently choose the operation to remove the tumor, and assist with radiotherapy and chemotherapy after operation. Though combined therapy have extended the average suivival time for maglinant glioma patients, the prognosis is not ideal. Recently, the immunotherapy of glioma is currently intensively investigated studied, which has acquired some achievements, but the properties of dendritic cell (i.e. DC) restrict the extensive application of DC vaccine in clinical field.Exosomes are found recently that they are nanometer sized membrane vesicles with typical cup-shape morphology. The vesicles separated from rat cortical neurones are at a density between 1.11 and 1.19 g/ml and have an average diameter of 100 nm. They are secreted into the extracellular milieu by a multitude of eukaryocytes as a consequence of fusion of multivesicular bodies (i.e. MVBs) with the plasma membrane. The early stages in exosome synthesis follow that of intralumenal vesicles, which form by inward budding of the endosome membrane, and during the formation of MVBs many cargos are sorting into the intraluminal vesicles. Once released, exosomes can fuse with membranes of other cells, delivering membrane and cytoplasmic proteins and mediating interaction between two cells without direct cell-to-cell contact.Exosomes can play critical roles in signal transduction and immune response. DEXs are highly enriched MHC class I and II complexes, costimulatory molecules, hsp 70 and hsp 90 chaperones. Through the MHC-peptide complexes they harbor ,DEXs could stimulate T cells directly. They could be captured by other professional APCs, which could amplify the effect of stimulating T cells. But their strong immunological activity depends on the maturation state of DC to prime MHC restricted and tumor antigen peptide specific cytotoxic T lymphocyte clone. TEXs bear not only MHC I, heat shock protein, tetraspanins but also tumor antigens. HSP could bind with many category antigen molecule to form a multiple antigen which could stimulate efficiently tumor antigen specific T cell immunological response. TEXs could present tumor antigen to T cell directly or isogenic DC to amplify the anti-tumor immunological effect by MHC class I molecule cross-presentation.The first exosome-based tumor vaccine Phase I clinical trial had been accomplished and detected that exosome vaccine have a number of superior qualities than DC vaccine. It highlighted the feasibility of large scale exosome production, long-term conservation, qulity control and the safety of exosome administration and high level immunogenicity and immunoreactivity. It is encouraging for the future development of exosomes in cancer immunotherapy.At present, the process of purification of exosomes from cell culture supernatants involves a series of differential centrifugations followed by three times high-speed ultracentrifugation at around 100,000×g for at least 1 h to pellet exosomes. And we need to prepare D2O/sucrose density cushion for a density gradient centrifugation. Many scholars only use differential centrifugation to isolate exosomes so as to lower the research and application technical threshold of exosomes.Our study is aiming at detecting whether glioma can secrete exosomes and exploring a simple and pragmatic methed to separate exosomes.Objective:To separate glioma-derived exosomes. To compare differential centrifugation with density gradient centrifugation.Method:1. First dissociate cells from fresh glioma of patient and use them for primary cultures of glioma cells.2. The second step is purify and isolate the exosomes from the glioma cells culture supernatant by both differential centrifugation and density gradient centrifugation respectively.3. Transmission electron microscopy is used to identify the two products in morphology. 4. To analyze whether the composition of their proteins are identical or not by using Sodium dodecyl sulphate PolyAcrylamide Gel Electrophoresis (i.e. SDS-PAGE).Result:1. The both group confirm that glioma could secrete exosomes which are similar in morphology. They are at a diameter between 55 and 155 nm and have an average diameter of 105 nm.2. We can confiem that the two products have almost the same protein profile by SDS-PAGE analyzing.Conclusion:1. We confirme that glioma could secrete exosomes which load a plenty of proteins related to tumor immunity.2. Comparing with the exosomes separated by classical density gradient centrifugation, the exosomes separated by differential centrifugation are similar in morphology and the composition of their proteins are identical. Thus it doesn't influence the finding about glioma in immunology. And differential centrifugation have lower research and application technical threshold to isolate exosomes.