| Malignant tumor is one of the threats of human health. At present, the conventional treatments are always inefficient. Biotherapy, especially tumor vaccine, has brought new hopes for human beings who have suffered a lot from rumors and have become one of the focuses of tumor treatment and studies. Methods employed to prepare vaccine include rumor cells genetically modified with cytokines, costimulatory molecules and tumor antigenic peptides, dendritic cells (DC) primed with tumor antigens in vitro or genetically modified with tumor antigens, or fusion of tumor cells with antigen presenting cells (APC). Though with various advantages, these tumor vaccines are because of complicated to prepare, restricted availability of relevant tumor antigens, the lack of molecularly defined tumor antigen delivery or targeting systems and the effects are relatively not promising.Exosomes are small membrane vesicles of endocytic origin that are secreted by various cells such as mast cells, T lymphocytes, B-lymphocytes, DCs and tumor cells. Tumor cell-derived exosomes containing tumor antigens and MHC class I molecule could present tumor antigens to DCs and induce CD8+T cell-dependent antitumor immune responses significantly. So they are as a novel efficient tumor vaccine to be used in antitumor immunity. This novel and efficient vaccine, with the non-cellular structure and no exogenous gene modification, is potential to be not toxic to the recipients. Together with characters that it is easy to prepare, this non-cellular vaccine is promising in clinic.Heat shock proteins (HSPs) are molecular chaperones with potent adjuvant capability in the induction of antigen-specific CTL, Thl response and increasing immunogenicity of tumor cells. It was reported that heat shock could induce tumor cells express HSPs, product chemokines that chemoactrract DC in vitro and induce potent antitumor immunity.In this study, we heat shocked A20 tumor cells, and then prepared heat shocked tumor cells-derived exosomes (HS-Exo). Our studies investigated the biogenesis, immune functions and effects of antitumor of HS-Exo, and also went further to study the related molecular mechanisms, providing a new clue for preparing highly efficient tumor vaccines. The findings will be described in two parts as follows.Part I: Preparation and biological functions of heat shocked-tumor cell-derived exosomes.Objective: To prepare heat shocked-tumor cell-derived exosomes and to identify the HS-Exo through morphologic observation by electron microscopy and SDS-PAGE. To detect the protein markers and molecules present in HS-Exo. To study the bioactivities and immune functions of HS-Exo in vitro experiments. And to find the differences between HS-Exo and Exo in several aspects including physical characters, bioactivities and functions.Methods: We isolated exosomes from supernatant of heat-shocked mouse B lymphoma cells A20. The A20 cells of heat-shocked group were exposed to 43 water bath for 1 hour, then recovery in 37 incubator for 4 hours. The culture supernatants of heat-shocked and control A20 cells were collected to isolate exosomes through a sequence of differential centrifugation steps. Briefly, cells were removed by centrifugation for 5 min at 300x gmax. Supernatants were collected and centrifuged (at 4 ) sequentially once for 20 min at 1,200 x gmax, once for 30 min at 10,000 x gmax and once for 60 min at 100,000 x gmax. And the resulting pellets were exosomes. Exosomeswere characterized through SDS-PAGE, immunoblot analysis of protein markers such as immune molecules and observation in transient electron microscope. The cytokines were detected using ELISA.We assessed the effect of HS-Exo on DC maturation. Immature DC generated from mouse bone marrow by culturing in GM-CSF and IL-4 for 5 days were incubated with HS-Exo or Exo for 48 hours, and then were collected for phenotypical analysis and phagocytosis of OVA-FITC by FACS technique. Cytokines in the culture supernatants were quantitated using ELISA. In the experiment of MLR to detect...
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