Extraction and differentiation of mature and immature dendritic cells from Lewis rat bone marrow / 中国组织工程研究

ABSTRACT

BACKGROUND:Dendritic cells exhibit extremely strong antigen phagocytic function in the immature stage,and they can demonstrate great advantages in immune tolerance,cancer immunotherapy,and other aspects.However,due to the extremely low content of immature dendritic cells in living organisms,its clinical and scientific applications are severely limited. OBJECTIVE:To study the extraction and identification of mature and immature dendritic cells from Lewis rat bone marrow. METHODS:Bone marrow precursor cells were isolated from the bone marrow of Lewis rats,and immature dendritic cells were induced by 20 ng/mL of granulocyte colony-stimulating factor and 10 ng/mL of interleukin-4 for 7 days,and then mature dendritic cells were induced by adding 1 μg/mL of lipopolysaccharide to immature dendritic cells for 2 days.The morphology of dendritic cells was observed using inverted fluorescence microscopy.The surface-specific molecules of mature and immature dendritic cells were identified by flow cytometry,and the secretion levels of supernatant interleukin-10,interleukin-12,and interleukin-17A in mature and immature dendritic cells were detected by ELISA.The response of mature and immature dendritic cells to T lymphocyte stimulation was measured by mixed lymphocyte reaction. RESULTS AND CONCLUSION:(1)The dendritic cells showed an obvious protrusion structure under an ordinary inverted fluorescence microscope.(2)Flow cytometry showed low expression of CD40,CD86,and other co-stimulatory molecules in immature dendritic cells.On the contrary,mature dendritic cells highly expressed the above co-stimulatory molecules.(3)The secretion of interleukin-10 and interleukin-17A in immature dendritic cells was much higher than that in mature dendritic cells(P<0.01).Interleukin-12 secretion in immature dendritic cells was much lower than that in mature dendritic cells(P<0.05).(4)Mature dendritic cells stimulated T cells significantly better than immature dendritic cells,and the stimulation ability was stronger when the ratio of mature dendritic cells to T lymphocytes reached 1:10.(5)The results indicate that Lewis rat bone marrow precursor cells can differentiate into dendritic cells and distinguish between mature and immature dendritic cells by flow cytometry identification,related factor detection,and mixed lymphocyte reaction.