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Efficient Induction of Antitumor Immunity by Exosomes Derived from Heat-shocked Tumor Cells

Author: ChenZuoLin
Tutor: CaoXueTao
School: Zhejiang University
Course: Oncology
Keywords: Exosomes Heat shock : A20 cells : antigen presentation Tumor immunity T cells Dendritic cells
CLC: R730.3
Type: PhD thesis
Year: 2004
Downloads: 151
Quote: 0
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Abstract

The malignancy serious threat to human health, the traditional treatment although a certain effect, but it is very limited. Biological treatment has brought new hope to cancer therapy, the tumor vaccine to become one of the hotspots of the cancer treatment. Existing method of preparation of tumor vaccines attention of various cytokines, costimulatory molecules, tumor antigen peptide gene-modified tumor cell vaccine; vitro antigen sensitized tumor antigen peptide gene modified dendritic cell vaccine ; tumor cells with antigen presenting cells of the fusion vaccine. The mechanism of these tumor vaccine prepared is to enhance the role of tumor antigen delivery was. The disadvantage is the complicated preparation process, the effect is relatively less than ideal. Exosomes are secreted by a variety of cells derived from a multi vesicle small membrane vesicles. Mast cells, T lymphocytes, B lymphocytes, dendritic cells (DC) and tumor cells can secrete Exosomes. Among them, the most attention is tumor cell sources Exosomes. Containing MHC Ⅰ molecules and LAMP-1, can be a tumor antigen presented to antigen presenting cells, produce significant CD8 ~~ T cell-dependent anti-tumor immune response. Suggest that tumor cell-derived Exosomes can be used as a new and efficient tumor vaccine for tumor immunotherapy. Indeed, Exosomes with non-cellular structures without genetically modified and exogenous gene into the body potential harmful effects, the preparation is simple and the advantages are everyone's attention, a new direction to study novel tumor biological therapeutic agents and tumor vaccines . Heat shock proteins are a large class of immune adjuvant effect chaperone, mainly induce antigen-specific cytotoxic T lymphocytes, Th1-type immune response and increase the immunogenicity of the tumor cells roar. The existing experiments confirmed that heat shock can induce the expression of heat shock protein, to promote tumor cells produce large amounts of chemotactic Zhejiang University Doctoral Dissertation Oncology factor, raising the DC local tumor infiltration by endogenous portable tumor antigen Zuo agent-like molecules to antigen effectively presenting, and induce the body to produce a strong anti-tumor immune response. The subject application A20 tumor cells, the role of heat shock, the preparation of heat shock tumor cell-derived Exosomes. Through the study of its protein composition, biological activity and immunological function and its anti-tumor effect, explore the immunological mechanism, hoping to provide new ideas for a new efficient anti-tumor effect of tumor vaccine. Preparation and biological properties of the first part of the heat shock tumor cell-derived Exosomes immunological function research purposes: to build heat shock tumor cell sources ExosomeS preparation system, and heat shock tumor cell-derived Exosomes morphological identification; detect heat shock of tumor cells sources ExoS0meS contains protein components: research the thermal shock source of tumor cells ExosomeS biological characteristics and immunological features; comparative heat shock tumor cell-derived Exosomes normal culture tumor cell sources ExosomeS, the protein composition of biological activity and immunological function on the similarities and differences. METHODS: We selected mouse B lymphoma cell line A20 tumor cells to create heat shock tumor cell-derived Exosomes preparation system. H heat shock conditions for 430C heat shock (water bath), placed in 37 ° C CO2 incubator recovery 4h charged culture supernatant. The classical four-step centrifugation, i.e. the 4 ℃ centrifugation 300 9 X10 minutes; l, 200 9 X 30 minutes; 10,0009 x 30 minutes; ultracentrifugation at 100,000 gx 60 minutes after isolated and purified Exosomes. Form of by TEM observations heat shock A20 tumor cells the source ExosomeS (Heat shoek of exosomes HS an Exo) and normal cultured the AZO tumor cell-derived Exosomes (Exo) by SDS 1 GE electrophoresis analysis Exo HS an Exo protein banding pattern composition to identify the source of tumor cells ExosomeS. A20 tumor cell sources Exo and HS immune molecules contains an Exo; detected by Western blot, ELISA method A20 tumor cell-derived Exo and HS Exo contained chemokines and cytokines. Study A20 tumor cell sources Exo and HS Exo DC cell immune FACS technology to detect relatively specific marker of DC cell surface molecules and a FITC OVA within swallow to observe Oncology Exo Zhejiang University Doctoral Dissertation 48h DC maturation state; processing and HS Exo Exo processing by the ELISA test Exo and HS DC cell culture supernatant proinflammatory cytokines and chemokine secretion; mixed lymphocyte reaction (MLR ) experimental observation HS an Exo affect the role of the DC cell immunostimulatory effects; the antigen sensitized CD4 T cells through the preparation of A20 tumor cells, A20 tumor cells the source EXo and the HS EX. Processing the DC cells detecting a Y level of the antigen sensitization CD4 T cell proliferation and secretion of workers FN to study DC cells in antigen presentation function and stimulate antigen-specific T cell activation ExosomeS treatment. Using of JH a TdR incorporation splenic T cells treated ExosomeS the observed the AZO tumor cell sources Exo and flS Ex. Proliferation of allogeneic splenic T cells. Result: the establishment of a heat shock tumor of cell sources ExosomeS the preparation system, stable and effectively isolated ExosomeS. We found that the different type of the total protein of the product was isolated with the A20 cell extract and cytosolic protein band in the overall protein band type. HS under the transmission electron microscope Exo morphology similar to Exo was SOnm 50 a double membrane vesicle-like structure, relatively uniform size, structural integrity. Protein detected culture the A20 cells A20 cells Exosomes derived from heat shock ExosomeS in both containing MHCI molecules derived from normal sources of like the LAMP and HSC7O, tumor cells ExosomeS characteristic protein. And HS Ex. MHCI molecules in content than Exo. HS Exo containing immune molecules involved in antigen presentation, MHC 11 molecules and CD86 molecules content significantly higher than Exo; the the HS Exo contain CD4O molecules in Exo not detected; neither detected CD80 molecule . Heat shock-induced cell expression of heat shock proteins, detection of the Exosomes heat shock protein found that there are HSP60, HSP70 and of HSP90 Exo Exo and HS HS Exo in HSP6O and HSP90 content was significantly higher than Exo of HSP70 of?

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