| Objective: To investigate the experimental conditions of culturing mouse bonemarrow-derived immature dendritic cells (imDCs) in vitro, and the feasibility onconstruction of tolerogenic dendritic cells (Tol-DCs) characterised by high IL-10expression induced by IL-10recombinanted lentivirus vector.Methods: The mononuclear cells were isolated from mouse bone marrow understerile condition in vitro. ImDCs were cultured and generated by low doses ofrecombinant mouse granulocyte and macrophage-colony stimulating factor (GM-CSF)and interleukin-4(IL-4) induction in vitro, then were purified with anti-CD11cantibody-coated magnetic beads. In order to induce into mature dendritic cells(mDCs), some of the imDCs were stimulated by tumor necrosis factor-alpha(TNF-alpha). The morphological changes of DCs were observed with light invertedmicroscope. The phenotypic differences between imDCs and mDCs were analyzed byflow cytometry. Mouse imDCs were transfected by IL-10recombinanted lentivirusvector, the green fluorescent protein (GFP) lentivirus transfected DCs positive controlgroup and untransfected DCs negative control group were designed at the same time.The expression of GFP were observed with fluorescence microscope and the level ofIL-10from supernatant solution were detected by ELISA in groups. The Tlymphocyte proliferation was observed by mixed lymphocyte reaction in IL-10lentivirus transfected imDCs group and untransfected imDCs, then the numbers ofregulatory T cells from cell mixture of the reactive cells vs stimulating cells ratio of1:10were detected by flow cytometry, the single T cells without DCs stimulatingnegative control group was added for purpose.Result: A large numbers of imDCs which had high purity were culturedfollowing the above methods and displayed the typical morphological characteristicsof DC over the culture time. The flow cytometry results showed that the imDCs hadnotably lower expression in MHCâ…¡, CD80, and CD86than mDCs. ImDCstransfected by IL-10recombinanted lentivirus vector had high expression of GFP with fluorescence microscope, and had higher expression of IL-10obviously thanother two groups detected by ELISA (P<0.001). The GFP transfected group and blankcontrol group all had low secretion of IL-10, there was no significant differencebetween them (P=0.997,>0.05). The capacity of IL-10transfected imDCs tostimulate the proliferation of T lymphocyte in vitro was weaker than that of naiveimDCs (P<0.01), but the proportion of regulatory T cells was higher than the latter(P<0.01).Conclusion: The mouse immature dendritic cells with low expression ofcostimulatory factors were obtained from bone marrow mononuclear cells induced bylow doses of GM-CSF and IL-4cytokines. The IL-10gene transducted dendritic cellshave efficient and stable expressions of IL-10induced by recombinanted lentivirusvector. Genetically modified immature dendritic cells hold the immune tolerancecharacteristics of promoting regulatory T cells growing and inhibiting T lymphocyteproliferation. |
