Effect Of Immunosuppressant On Differentiation And Maturation Of Murine Bone Marrow-Derived Dendritic Cells

OBJECTIVE To establish the Operation Procedure(OP) of dendritic cells cultured in vitro by marrow cells of mice and obtain high purity immature and mature dendritic cells. To observe the different effect of several immunosuppressants on dendritic cells in different mature status in vitro.METHODS Dendritic cells were cultured in vitro by marrow cells of mice ,and were divided into six groups: group A was added stimulated factors including rrGM-CSF,rrIL-4,rrTNF-α,rrIFN-γ; besides the stimulated factors above, group B,C,D,E,F were added different immunosuppressants (DEX,CTX and MP) with different dose on the 6th day. To observe the different mature status of dendritic cells in the group. Level of IL-12 in the culture medium was assessed by ELISA, and surface costimulatory factor CD80,CD86 of dendritic cells of the six groups were assessed by flow cytometry .RESULTS①Culture of dendritic cells : Growth of precursor in bone marrow was good in medium plate under invert microscope. A large number of cells was proliferation, and harvestes on the 6th day and 9th day. 2.0-5.0×10~7 dendritic cells were obtained in marrow cells of every 2 mice, which could meet the requirement of experiment.②The different characters could be found in immature dendritic cells.③The level of IL-12 increased with the increase of culture day.④DC differentiated in the presence of drugs had decreased level of IL-12(p<0.05).⑤The expression of CD80 and CD86 of mature DC was significantly higher than those of immature DC.⑥DC differentiated in the presence of immunosuppressants had decreased expression of CD80 and CD86(p<0.05).⑦There were no significant difference observed in multiple comparison among presence one and two immunosuppressants group.CONCLUSION we successed establish the Operation Procedure(OP) of dendritic cells cultured in vitro by marrow cells of mice and obtain immature and mature dendritic cells. DEX,CTX and MP have the inhibitory effect on DC mature . The inhibitory effect in higher does groups is higher than that of lower dose one. It shows that the presence of two immunosuppressants does not express a higher inhibitory effect.