Research On Prevention And Treat Graft Versus Host Disease By Donor Immature Dendritic Cells

Objective: Based on the generated mechanism for graft-versus-host disease (GVHD), establishing the modulating model of dendritic cell induced lymphocytes in vitro, to observe the ability of immature dendritic cells that induce the stimulation of autogeneic naive T cells proliferation, to investigate the feasibility of clinical application that immature dendritic cells prevent and treat GVHD.Methods: Mononuclear cells were isolated from the peripheral blood of the healthy donor. They have been induced and differentiated into immature; dendritic cells with rhGM-CSF and IL-4 for 5 days; and induced for 7 days, and then doped LPS and dendritic cells have been differentiated into mature dendritic cells for 18 hours. Then the morphology of the cells was examined through inverted microscope and hematoxylin and eosin stain, their immunological phenotype CD86 and HLA-DR that belong to MHC- II were examined with immunohistochemistry stainning; CDla and CD83 were examined with flow cytomcter. According to the method of mixed lymphocyte reaction (MLR),constructed the model of the generated mechanism for GVHD,we set up three experimental groups: control group, imDC group and mDC group. Dendritic cells and MLR have co-cultured for 48 hours, doped cell counting kil-8(CCK-8) to detect optical density, to compare the ability of donor immature and mature dendritic cells that induced the stimulation of auto-allergic naive T lymphocyte proliferation.Results: (l)The cells exhibited typical morphology of imDC after 5 days, which semi-floated into medium and have many shorter sentus around cell. The shape of cells is various and the cells are bigger than Mononuclearcells. Each of the specific expression of CDla CD83 and double antibody are (67.06?.93)% (66.82?.06)% and (62.34?.94)%, with lower mature expression CD83 was detected by flow cytometry; and the cells exhibited the typical morphology of mDC after 8 days, which float in medium and have many long edge hair. The cells are rounder and bigger. Each of the specific expression of CDla CD83 and double antibody are (64.98?.99)% (86.44?.10)% and (65.16?.55)%, with higher mature expression CD83; (2)dendritic cells and MLR have co-cultured for 48 hours, and doped cell counting kit-8(CCK-8) to detect optical density, statistical analysis showed that there was no obvious difference between imDC and control group(P>0.05). However, mDC group had significant difference that compared with both imDC and control group (P<0.01), and the stimulation index that the autogeneic T cells was stimulated is more than 2.00{S1=2.22), there was significant difference that compared with 2.00.Conclusion: According to the result of morphology, immunohistochemistry and the cells' immunological phenotype was detected with flow cytometer, we can draw a conclusion that we have successfully obtained imDC and mDC with rhGM-CSF+IL-4 induction and cultivation; In the modulating model of dendritic cell induced lymphocytes in vitro, the result that CCK-8 detected hint that donor imDC is capable to induce autogeneic T cells to have low reaction, and it maybe become a method to prevent and treat graft versus host disease.