Empirical Study Of Inducing Murine Bone Marrow Cells To Immature Dendritic Cells In Vitro

Objective:To establish an effective and convenient method in vitro for induction and amplification of immature dendritic cells(imDC) by using mouse bone marrow cells as the precursor cells.Then it will lay the foundation for the experimental study and clinical application of imDC.Methods:The experiment was divided into three groups: GM/IL-5,GM/IL-7 and GM/IL-LPS; Firstly, preparating C57BL / 6 mouse bone marrow cells, then cracking erythrocytes with Tris-ammonium chloride buffer(Tris-NH4Cl); Secondly, the bone marrow cells were cultured with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin-4(rmIL-4) in vitro, and the floating granulocyte and lymphocyte were wiped off after incubuting 48 h. We have changed the half-quantity medium once in other days since the second day to ensure the concentration of cell factor;Finally, according to the characteristic that DC separated from plastic after training 72 h automatically, the suspending and loosely adherent cells were collected on the fifth(GM/IL-5)and seventh day(GM/IL-7)respectively.As to GM/IL-LPS, the suspending and loosely adherent cells were collected after adding large dose of lipopolysaccharide (LPS) on the sixth day for further cultivating 18 h. To identificate the morphology and biological function of the three group cells,we used scanning electronic microscope observing the morphology,flow cytometry (FCM)examining the expression of phenotype molecular and allogeneic mixed lymphocyte reaction (MLR) detecting three group cells to stimulate allograft T cell proliferation.Results: The three group cells exhibited typical morphological characteristics of DC under scanning electron microscope and had a much higher CD11c expression rate by flow cytometry, which was over 73%.Otherwise, GM/IL-5 DC had a lower expression rate of CD40,CD86,MHC-â…¡,which was 34.46%,34.21%,45.16% , that in GM/ IL-7 DC was 46.99%,68.79%,94.37%,and that in GM/ IL-LPS was 78.68%,89.84%,96.75%, respectively.In MLR, GM/IL-5 DC had the weaker ability for stimulating the proliferation of allogenetic T cell compared with GM/ IL-7 and GM/ IL-LPS DC(P<0.05).Conclusion: The immature dendritic cells,which had typical morphological characteristics of DC,cellular phenotype and high purity, could be induced and amplified from mouse bone marrow by using the method; Whatsmore, it could not only reduce the cost and shorten the time, but also can define the differentiation time between immature and mature dendritic cells. Therefore, it will lay foundation for the further study on the clinical application of imDC inducing immunological tolerance after organ transplantation.