| Malignant tumor is one of the threats of human health. Among the diseases causing death, tumor has climbed to be the one of the major killer and is still becoming more and more imposing. 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 tumors and have become one of the focuses of tumor treatment and studies. Methods employed to prepare vaccine at present include tumor cells modified with cytokines, costimulatory molecules and tumor antigenic peptides, dendritic cells (DC) primed with tumor antigens in vitro or genetically modified with tumor antigens and the fusion of tumor cells with antigen presenting cells (APC). Though with various advantages, these vaccines are always complicated to prepare and the effects are relatively not promising.During the study of the relationship between heat shock protein (HSP) and tumor, we found that after heat shock, culture supernatants of tumor cells could chemoattract DC and T lymphocytes. And we went further verify that chemokines were present in the culture supernatants of tumor cells after heat shock. Because of the rapid presence of this activity, we hypothesis that chemokines be released out of tumor cells through a particular secretory form. Our further studies showed that this kind of exocytosis form is exosome as we detected various chemokines in the exosomes derived from tumor cells after heat shock and no chemokines were detected in the supernatants after isolation of exosomes. We also found that abundant MHC molecules, costimulatory molecules, adhesion molecules and chaparones carrying tumor antigens. As the exosomes are derived from tumor cells, carry tumor antigens and sharecharacters with most of the vaccines prepared previously, they are a kind of non-cellular novel vaccine. In vivo experiments showed that this kind of novel vaccine could not only protect mice from syngeneic and allogeneic tumor challenge but inhibit the growth of established tumors. This novel and efficient vaccine, with the non-cellular structure and contained no exogenous gene modification, is potentially no harm to the recipients. Together with the characters that it cause no ethical problems and it is easy to prepare, this non-cellular vaccine is promising and economic both in clinic and in market.Heat shock and hsp70 can stimulate tumor cell produce chemokines that can chemoattract DC and T lymphocytes in vitro.Tumor cell culture supernatant after heat shock could chemoattract DC and T cells, and hsp70 was abundantly present in the supernatants. After heat shock of human and mouse cell lines A549, LoVo, HeLa, MCF7, 3LL, CT26, B16, NIH3T3, TC-1 and 107B for Ih at 43癈 and recovery in 37癈 incubator for 4h, the culture supernatants can chemoattract DC and T cells in an in vitro chemotactic assay, indicating that chemotactic products are present in the supernatants. Considering that various cell lines demonstrate the same kind of chemotactic activity, it can be concluded that this effect is a kind of universal phenomenon. As it has been reported that HSP70 can stimulate CD8+ T cells to produce chemokines, we stimulate tumor cells 3LL with HSP70 (1 u g/ml) for 4 h and collect the culture supernatants to perform in vitro chemotaxis assay. We found that the supernatants can also chemoattract DC and T cells. To exclude the possibility that HSP70 can chemoattract DC and T cells directly, we add HSP70 to the lower well and we found no chemoattraction happened. Thus HSP70 can stimulate the chemoattracting product to produce. As chemokines are the major proteins inducing cell migration, we examined the mRNA expression in 3LL cells after heat shock or HSP70 stimulation and proteinexpression in 3LL culture supernatants by RT-PCR and Western blot respectively. We found that, after heat shock and HSP70 stimulation, various chemokines are upregulated at mRNA level in 3LL cells and at protein level in the culture supernatants. HSP70 alone can't completely substitute for h...
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