| The IRPS which is isolated and purified from Radix is an avtive ingredient which possesses antivirus,antioxidation and immunoregulation,confirmed by many researchers. Dendritic cells are the most powerful professional APCs with the unique features of inducing primary immune response, stimulating the proliferation of naive T cell and regulating of cellular and humoral immunity. In addition, DC also is one of target cells of plant polysaccharides.To investigate the effects of IRPS on DC, bone marrow-derived hematopoietic stem cells,were isolated from mouse and induced their differentiation into DC subsets with different cytokines. Then IRPS were added to the different DC subgroup,and the effects of IRPS were evaluated by cell proliferation,maturation,secretion of cytokine and allogeneic mixed lymphocyte reaction.1. Inducing dendritic cells (DC) from bone marrow (BM) cellAfter collecting bone marrow cells from mouse and purifying them by adherent method, the DCs were induced in following three different conditions. DCo were induced by culture of BM cells (107cells/well) in the presence of GM-CSF (20ng/ml) and IL-3(20ng/ml) in a Costar12-well plate. DC1were induced by culture of BM cells in the presence of IFN-γ (20ng/ml) and IL-12(20U/ml) in addition to GM-CSF plus IL-3. DC2were induced by culture of BM cells in the presence of IL-4(20ng/ml) in addition to GM-CSF plus IL-3. DC culture medium with fresh cytokine were exchanged in half in every other day for7days. DC subsets induced by different cytokine are all the typical dendritic cells by cellular morphology with microscope and scanning electron microscope.2. Determing optimal concentrations of IRPS on DCs subsets proliferation by CCK-8assayAmong the range of testing concentrations from0.025ug/ml-3.2ug/ml, IRPS can stimulate the DCs proliferation on different level. Concentration of IRPS boosting DCs proliferation is0.4ug/ml.3. Effect of IRPS on DCs morphologyThe DC subsets treated with IRPS showed more protrusions and rougher surface in morphology than the untreated DCs by scanning electron microscope.4. Mixed lymphocyte reactions (MLR)BM derived DCs collected as stimulating cell, lymphocyte isolated from allogeneic mouse spleen cells as responders were mixed with DCs in the ratio of1:6.6and cuitured for3days.The result of experiment showed that different DC subsets all have strong stimulant ability for the proliferation of lymphocytes, and after the treated with IRPS, DCo,DC1and DC2subsets all can significantly improve the proliferation of lymphocytes. 5. Luminex xMAP assay for cytokines of DC subsetsSupernatants of mature DC subtypes cultures were collected for detecting IL-12,IFN-γ,IL-10and IL-4, using Milliplex kit with Luminex., DC1subsets secreted more IL-12compared with DCo and DC2,after treatment with IRPS, the level of IL-12in DCo and DC2significantly reduced, and IL-4in culture supernatant of DC2subsets also significantly reduced, there was no significant differences on the level of IFN-γ, thus we guess IRPS inhibit Th1cytokine IL-12produced by DC0,DC2subsets,and boost Th2cytokine IL-4produced by DC2.6.Luminex xMAP assay for cytokines of Mixed lymphocyte reactionsAfter the treatment with IRPS, DC1showed a polarized supporting effect on the differentiation of Thl cytokine IL-12and IFN-γ, promoting lymphocyte differentiation to Th1. While cytokine of IL-4produced by DC2stimulating lymphocyte was significantly reduced.7. Flow cytometry (FCM)assay for Cell surface marker analysis of DC subsets by IRPSThe phenotypic characterization of DC subsets were detected using FCM. The DC0、DC1and DC2subsets were no significant differences in the expression of CD11c, but when they were treated with IRPS, all of them had been obviously improved in the expression level of CD11c、MHC-Ⅱ and CD80.DC1subsets expressed the highest levels of MHC-Ⅱ、CD80and CD86molecules compared with DC0and DC2, although no significant difference was observed in CD54expression. |
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