Effect Of Trichostatin A On Phenotype And Function Of Mouse Bone Marrow-derived Dendritic Cells

Objective： The immature dendritic cells can induce immunological tolerance andit’s that how to maintain its immature state becomes research hotspot. To investigatethe effect of Trichostatin A on on phenotype and function of mouse bone marrow-derived dendritic cells.Methods：Mice myeloid dendritic cells isolated from mouse bone marrow wereinduced, cultured and amplified in complete medium with recombinant mousegranulocyte-macrophage colony-stimulating factor (10ng/ml) and recombinant mouseinterleukin4(10ng/ml).When culturing for6days, cells were divided into four groupsrandomly: control group(without any factors),TSA group(treated with TSA100ng/ml), lipopolysaccharide group(treated with LPS100ng/ml),LPS+TSA group andculturing for48hour. Cell morphology was observed under inverted microscopeeveryday.On the eighth day the cultivating dendritic cells in the four groups weretaken to adjust the cell concentration of5×106/ml after washing in PBSrespectively.100μl suspension was put into the testtube for cytometry,0.5ug antibodyof FITC-CD86, FITC-CD80was added. Flow cytometry was used to detect cellphenotype after washing with PBS again. Stimulating activity for allo-genetic spleniccells were assessed with MTT on the eighth day respectively. IL-10and IL-12concentration in dendntic cells supernatant were measured with ELISA(enzymelinked immunosorbent assay) on the eighth day.Results: Mouse bone marrow derived dendritic cells were observed under invertedmicroscope. Most cells appeared round in shape and adhered to the tissue cultureplastic plate. With the extension of the culture time, some appeared markedlyenlarged in shape and had an anomalous appearance. After two days, the cellsscattered single cells gathered in fascicular cell colonies. Five days later the cellcolonies grew and increased. When culturing for6days, the cell colonies appeared many branches or pseudopodia-like formations around on edge. The shape of the cellssuggested a typical dendritic cells morphology; Compared with the other group, themembrane expression of CD86and CD80levels on dendritic cells was significantlydecreased after treatment with TSA (P <0.05).In mixed lymphocyte reactions,TSA-treated dendritic cells showed less T lymphocytes stimulatory capacity.Investigated the effect of TSA on the secretion activity of dendritic cells. We foundthat TSA could enhance the secretion level of the Th2related cytokineIL-10(P<0.05),but inhibit significantly the secretion level of the Th1related cytokineIL-12（P<0.05）.Conclusions: TSAcan inhibit the maturation of dendritic cells and induce productionof the immature dendritic cells.The immature dendritic cells can not be activated bymaturation promoting factor，so which is potential to apply on the induction of immunological tolerance after autoimmune diseases and organ transplant.