Regulation On Phenotypic And Functional Maturation Of Dendritic Cells By Agaricus Blazei Polysaccharides And Sodium Selenite

Background and ObjectiveAgaricus blazed Murill(ABM) is an edible mushroom native to Brazil that is consumed as food for its nutritional and medicinal properties. It was discovered in 1960 by Takatoshi Furumoto a grower and researcher who sent it to Japan for investigation. Active metabolites can be isolated from fruiting bodies and pure culture mycelia, such as active polysaccharides, active nucleinic acid, active sterin , lectins and so on. As the major effective ingredient, previous experiment indicated thatAgaricus blazei polysaccharides(ABP )had anti-tumor, immunoregulation and antimutagenic properties. The major fraction of ABP being FIII-2-b, which comprised a protein complex composed of 43.4% protein and 50.2% carbohydrates.Se is an essential trace element of human which was discovered by Sweden scholar Berzelius in 1817. It has an extensive biologicalfunction, especially it can inhibit the growth of tumor cells.Dendritic cells (DC) are professional antigen-presenting cells (APC) playing key roles in innate and adaptive immunity. The property distinguishes them from all other APC is that it bearing sole responsibility for the stimulation of virgin T lymphocytes. So dendritic cells are forerunner of the immune reaction. Dendritic cells can uptake the antigen and present the antigen-MHC compounds to specific T cells, Then activate CD4~+T and CD8~+ lymphocytes which play important roles in antitumor immune response.The antitumor property of ABP is chiefly due to an immune reaction, involving activiation of macrophages, nature killer cells and T lymphocytes, and the release of interferon, all of which straight or indirectly influence the tumor cells. The anticancer capability of Se may be related to depressing the mutagenicity of the carcinogenic factors, inducing apoptosis, regulating GSH-Px, preventing and repairing DNA damage, regulating immune system and so on. But little is known about their immunomodulating effects on dendritic cells (DC).The present study is designed to observe the regulation on the phenotypic and functional maturation of dendritic cells derived from murine bone-marrow by ABP and sodium selenite in vitro, to elucidate the antitumor mechanism of ABP and sodium selenite related to DC, to provide the experiment basis for clinical application.Methods1. Murine bone marrow-derived dendritic cells culture: The mice were killed and their bone marrow cells from the femur and tibiae were flushed and depleted of RBC by hypotonic lysis using RBC lysing buffer. The cells were grown at a starting concentration of 1×10~6 cells/ml in RPMI 1640, supplemented with 10% fetal calf serum. Ten-nogram-per-milliliter recombinant mouse (rm) GM-CSF and five-nogram-per-milliliter IL-4 were added to the culture medium. We replaced the culture medium on alternate days.2. Different dosage of ABP and sodium selenite were added to the culture medium on the 3rd day. NS were added to the control group.3. BM-derived murine DC were harvested on the 6th day, observed using inverted microscope to determine the morphologic change. On the 8th day, the expression of CD86 and CD11a molecules were analyzed using a flow cytometer to evaluate the effect on differentiation of DC by ABP and sodium selenite.4. BM-derived murine DC were harvested on the 8th day, In order to evaluate the influence on the proliferation of DC by ABP and sodium selenite ,we detected the cell life cycle and DNA content by flow cytometry to analyse the proliferation of DC.5. On the 8th day, BM-derived murine DC were treated with 25μg/ml mitomycin C for 4h and mixed with allogeneic T cells from spleen for 96h, we detected the cell life cycle and DNA content of T cells by flow cytometry to analyze the proliferation of T lymphocytes.6. On the 3rd day, dendritic cells were loaded by the tumour antigen peptide of ascite hepatoma H22 cells. On the 8th day, BM-derived murine DC were harvested and mixed with allogeneic T cells from spleen for 48h, Then ascite hepatoma H22 cells were added to. 28 hours later, killing capabilities of allogeneic T cells were examined with MTT method.7. The results were expressed as the means±SD of the indicated number of experiments, and analyzed by statistical software SPSS 13.0, One-way ANOVA methods were used among the experimental groups. P-value of <0.05 was considered to be statistically significant.Results1. On the 6th day, Most of the BM-derived murine cells which treated with ABP waxed and presented the character of mature DC. The cells in the control group displayed typical characteristics of immature DC. We could not see obvious ecphyma of DC and the density of DC decreased in the sodium selenite group.2. The expression of co-stimulatory molecules CD86 and CD11a on DC surface increased in the ABP (50μg/ml, 100μg/ml, 200μg/ml) group, and the expression of them decreased in the sodium selenite group. But the expression of co-stimulatory molecules significantly increased in the ABP (100μg/ml) and sodium selenite group.3. ABP (100μg/ml) increased the proliferation index of DC .There was no significant difference between the sodium selenite group and the control group.But the proliferation index of DC decreased in the ABP (100μg/ml) and sodium selenite group.4. ABP (50μg/ml, 100μg/ml,) increased the proliferation index of T lymphocytes. Sodium selenite depressed it. The proliferation index of T lymphocytes significantly increased in the ABP (100μg/ml) and sodium selenite group. 5. Both ABP (50μg/ml, 100μg/ml, 200μg/ml) and sodium selenite could advance the killing capabilities of allogeneic T cells to ascite hepatoma H22 cells separately. Compared with the control group, there was no obvious elevation of the killing capabilities in the ABP (100μg/ml) and sodium selenite group.Conclusions1. ABP can enhance the maturation and differentiation of bone marrow-derived DC culture in vitro, promote the expression of the co-stimulate molecules, and this effect is facilitated by sodium selenite. Sodium selenite alone can inhibit the expression of the co-stimulate molecules.2. ABP can increase the proliferation of marrow-derived DC. Sodium selenite attenuated the effect of ABP to DC.3. ABP can promote the proliferation of T lymphocytes which sensitized by DC, and this effect can be facilitated by Sodium selenite.4. Both ABP and sodium selenite can promote the CTL cytotoxity against tumor cells separately. When together with ABP, Sodium selenite decreases the effect of ABP.