Influences on induction and maturation of mouse bone marrow-derived dendritic cells by varying combinations of cytokines / 中华微生物学和免疫学杂志

ABSTRACT

Objective To investigate the influences of culture conditions on the in vitro induction and maturation of dendritic cells by using different combinations of cytokines. -ethods Mouse bone mar-row cells were isolated and cultured in media containing varying combinations of cytokines, including granu-locyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and fms-like tyrosine kinase 3 ligand (Flt3L). After cultured at 37℃ for seven days, the attached bone marrow cells were collected and stained by fluorescence-labeled monoclonal antibodies (McAb) against CD11c, MHCⅡ and CD86 for flow cytometry analysis. In the parallel group, LPS was added on day 5 to a final concentration of 1μg/ml for DC maturation analysis by flow cytometry. Results In group Flt3L (20 ng/ml)/GM-CSF (20 ng/ml)/IL-4 (10 ng/ml), 90％ of bone marrow cells were CD11c-positive. Flt3L (100 ng/ml) could induce 88％ of bone marrow cells to express CD11c. Bone marrow cells positive for MHCⅡ accounted for 35. 4％ and 36. 1％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF, where both Flt3L and GM-CSF were used at a concentration of 20 ng/ml. After LPS stimulation, the positive rates of MHCⅡ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF were 58. 1％ and 59. 6％, which increased by 22. 7％ and 23. 5％, re-spectively. The percentages of CD86-positive bone marrow cells were 7. 1％ and 5. 5％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF. Bone marrow cells positive for CD86 grew by 7. 1％ and 6. 2％ in group Flt3L (20 ng/ml) and group GM-CSF/IL-4 after LPS stimulation. Conclusions Flt3L and GM-CSF probably dominated the differentiation and maturation of bone marrow-derived dendritic cells with a synergis-tic effect. Combined usage of Flt3L and GM-CSF at the concentration of 20 ng/ml would be an optimal proto-col for DC research.