| Objective:In order to verify the assumption that MSC induced mDC into DCreg through Wnt/β-catenin signaling pathway.Methods:(1) GM-CSF and IL-4 were added to induce bone marrow mononuclear cells differentiate into imDC. ImDC was stimulated with LPS for 48 hours to induce the generation of mDC. Using flow cytometry to detect DC’s surface markers and identifying its functions.(2) It were divided into mDC group, MSC+mDC group:mDC and MSC were co-cultured for 15 days, MSC+mDC+DKKl group:when mDC and MSC were co-cultured, Dickkopf-1 (DKK1) was added at day 1、3、 7、10、13. Dickkopf-1 (DKK1) is the specific inhibitor of canonical Wnt signaling pathway, which can inhibit the formation of Wnt-Frizzled-LRP6 ternary complex on DC cells. At day 15 collect supernatant and cells。(3) Detecting experiment indicators:① Detecting DC phenotype and function:a) flow cytometry to detect cell surface molecule CD11b, CD11c, MHCⅡ, CD40, CD86, and B220; b) fluorescence intensity to assesse the ability of DC phagocyte ovalbumin-FITC; c) Elisa assay supernatant the concentrations of inflammatory cytokines IL-10, IL-12 and TGF-β; d) using CCK8 to detecte the ability of DC stimulating CD4+T cell proliferation after they co-cultured for 5 days; ② Detecting Wnt signaling pathway associated protein in the DC: Western blot assay protein concentration of cytoplasm GSK and nucleus β-catenin in the DC.Results:(1) Successfully induced bone marrow-derived DC:Compared with imDC, mDC cells indicates a dendritic projections, and high expression of CDllc, MHCII, CD86, CD40, low expression of myeloid markers CD11b, B220, decreased endocytic activity (p<0.01), increased the secretion of pro-inflammatory cytokines IL-12 (p<0.05)and inhibition inflammatory cytokine TGF-β,IL-10 (p<0.01). It indicated that after bone marrow-derived mononuclear cells induced to mature DC, the phenotype and function of mDC was in line with DC in vivo migration process.(2) MSC is capable of inducing the differentiation mDC to DCreg:DCreg, which low expression of MHCⅡ and co-stimulatory molecules, have low immunogenicity and decreased ability to stimulate T cell proliferation while inhibiting an increase in secretion of inflammatory cytokines, possess low immunogenicity. Compared with mDC, the group of MSC+mDC (namely co-culture group) is high expression of CDllb, B220 and lower expression CD11c,MHCⅡ molecules as well as costimulatory molecules CD86, CD40 and increased the phagocytosis ability of OVA-FITC, the secretion of inhibiting inflammatory cytokines IL-10, TGF-β and reduced the inflammatory cytokines IL-12 and the ability to stimulate CD4+T lymphocyte proliferation.(3) The classical Wnt signaling associated protein expression in DC:compared with the mDC, the ratio of p-GSK/t-GSK in cytoplasm (p<0.01) and β-catenin in nuclear (p<0.05) was increased in co-culture group. It indicates that the classical Wnt signaling pathway is involved this cell differentiation process.(4) Suppression of the canonical Wnt signaling pathway DC can inhibit the differentiation of DCreg:after treatment of co-culture cells with Wnt signaling antagonist DKK1, compared with co-culture group, the ratio of p-GSK/t-GSK in cytoplasm (p<0.05) and β-catenin in nuclear(p<0.05) was decreased in the group of co-culture suppression group. Compared with co-culture group, co-culture suppression group is low expression of CD11b, B220 and higher expression CD11c, MHCⅡ molecules as well as co-stimulatory molecules CD86, CD40 and decreased the secretion of inhibiting inflammatory cytokines IL-10, TGF-β and increased the inflammatory cytokines IL-12 and the ability to stimulate CD4+T lymphocyte proliferation. It further indicates that the classical Wnt signaling pathway is involved this cell differentiation process.Conclusions:(1) MSC can induce the differentiation of mDC into DCreg.(2) The classical Wnt signaling pathway is involved this cell differentiation process. |
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