In Vitro Studies On Dendritic Cells Derived From Human Cord Blood And Its Immunological Capacity

ObjectivesThrough the proliferation of dentritic cells derived from human cord blood progenitor cells in vitro,DC were confirmed by morphology ,immune phenotype and to investigate the cytolytic activity of cytotoxic T lymphocytes (CTL) induced by DC.MethodsCord blood was collected under steril condition and the mononuclear cells (MNC) were separated by centrifugal in density gradient .The adhesion cells,collected among the MNC ,were divided into seven groups ,add 3 ml RPMI-1640 nutrient medium to every group, then add different combinated cytokines to induce the generation of DC.One of these groups is control group, not adding any cytokines. The last were added rhIL-4 add rhGM-CSF respectively.And three of them were added TNF-αon fifth day.After culture them for 14 days, collect adherent cells ,detect CD86,CD83, CD14, HLA-DR and other immune factors with flow cytometer.The capacity of DC to active allo-T cell depent and cells of the control allo-T cells anti- K562 tumor cells immnue responses was tested by MTT. While rate of target cell to effector cell is 10:1,the citotoxic activities is the highest (p<0.01),and the ratio is 28.97±6.62%.It shows the higher rate of target cell to effector cell,the higher citotoxic activities .ResultsAfter cultured for 14 days, the content of DC in group 6 (1000u/ml rhIL-4 and 1000u/ml rhGM-CSF,add TNF-α50u/ml on fifth day)is the highest,P<0.05 compared with other groups. Detect the number of positive DC with special antigens CD86,CD83,CD14, HLA-DR with flow cytometer, group 6?5?4?3?2?1?7. T cell activated by DC showed magnificent cytotoxicity on K562 tumor cells, p< 0.05 compared with control group.ConclusionsMNC from cord blood can be induced and differentiated into DC by combinated cytokines in vitro, after being cultured for 5 days in the culture medium of 1000U/ml rhIL-4 and 1000U/ml rhGM-CSF,add 50U/ml TNF-αto the medium, the number of DC induced is the highest. rhIL-4,rhGM-CSF and TNF-αcan not only promote the formation of cellular morphology, but also increase obviously the number of DC.CTL derived from antologous T lymphocytes induced dendritic cells possessed specific cytolytic effects against K562 tumor cells .