ABSTRACT
Objective To investigatethe role of NF-κB played in the process of the cord blood monocytes differentiating into dendritic cells(DCs)induced by astragalus polysaccharide(APS)and to explore the signal transduction pathway involved in this process.Methods Umbilica]cord blood was collected in aseptic conditions.The cord blood monocytes were obtained by density gradient centrifugation and were divided into three groups afterwards.In the control group.cells were cultivated in the RPMI 1640 complete medium.In the APS group.cells were cultivated in the RPMI 1640 complete medium containing 100 mg/L APS.In the PDTC group:cells were treated with 10 μmol/L disulfide carbamate(PDTC).NF-κB inhibitor in 30 min followed by cultivalion in the RPMI 1640 complete medium containing 100 mg/L APS.,The morphological changes were observed during the process of cultivation by the optical microscope and transmission electron microscopy.Cells were collected 12 d later and the cellular immunophenotyping was assayed by FCM.,The activation and migration of NF-κB fluorescence in the cells was examined by the immunoflouresce microscopy.Results (1)Cells in the control group grown up without cluster forformation and were found fusiform and macrophage-like in 12 d.Cells in the APS group grown up in clnstem,and morphological changes were found from the circular shape to a typical dendritic cells-like shape.Cells in the inhibitor group grown up slowly and without cluster formation,and cell morphdogy had no significant change.(2)The expression of DCs-specific antigen CD80,CD83 and CD86 in the APS group was higher than that in the control group and inhibitom group(P0.05).(3)NF-κB fluorescence in the nuclei was examined by the immunoflourescence microscopy and was much higher in the APS group than that in khe other groups,especially in 72 h with the activation rate of NF-κB (75.20±7.37)%,while(13.20±3.46)% of PDTC group and(8.20 ±1.92)%,respectively(P<0.01).Conclusion Astragalus polysaccharide can induce the differentiation of umbilical cord blood cells into DCs,and NF-κB is the key component of the signal transduction pathway involved in this process.
ABSTRACT
Objectives To elucidate the effect of APS replacing cytokine on inducing the cord blood monocyte in vitro into the dendritic cells (DCs) and its cellar immunological characteristic. Methods The cord blood monocytes were isolated and obtained by lymphocyte isolation. three groups were divided: ②Cultured in the RPMI-1640 culture with GM-CSF/IL-4/TNF-?,as the positive control group, with APS in concentration (100mg/L) as the experimental group,and without GM-CSF/IL-4/ TNF-?and APS,as the negative control group, respectively. The morphotype of DCs was identified by inverted optical microscope or scanning electron microscope. The phenotype of cultured 12 days DCs (CD1a, CD80, CD86, and CD83) was identified by flowcytometry. Results Cultured for 72 hours , the morphous of cell of the experimental group grew clustering and began to change from round to irregularity, appearing rough cell face and barb pustute. The longer cell cultured, the more obvious the dendritic structure is. The experimental group cell cultured for 12 days had the most typical dendritic structure. the negative control group cell had no dendritic structure and became the macrophage when cultured for 12 days. The experimental group cell cultured for 10 days showed typical dendritic morphotype by SEM. The experiment group cell and the positive group cell cultured for 12 days significantly expressed the high level phenotype of DCs((CD1a, CD80, CD86, and CD83))by flowcytometry. Conclusions APS and cytokine both could induce the cord blood monocyte to direofive differentiate into functional DC.
ABSTRACT
Objective:To observe the effect of Astragalus polysaccharides(APS) on inducing the cord blood monocytes into mature dendritic cells(DCs) in vitro and to investigate their morphous,cellar immunological characteristics,and contribution to T cell proliferation.Methods:①The cord blood monocytes were isolated by lymphocyte isolating solution under axenic condition,and three groups were divided.②Cells cultured with APS in concentration of 100 mg/L as the experiment group, that with the cytokines of GM-CSF/IL-4/TNF-? as the positive control, and another without either GM-CSF/IL-4/TNF-? or APS as the negative control, respectively. The morphology of DCs was identified by inverted optical microscope or scanning electron microscope. The phenotype of 12 days cultures of DCs(CD1a, CD80, CD86 and CD83) were identified by flowcytometry. The DCs preparations from the experiment group were treated with mitomycin for 45 min to remove their proliferative activity as incentive cells in the mixed cultures with allogenic peripheral blood mononuclear cells from healthy volunteers as responders. T cell proliferation induced with the DCs preparations was detected by MTT chromatometry.Results:After cultured for 72 hours, the cell of both the experiment group and the positive control grew dusteringly and began to change from round to irregularin shapes. The longer the cells were cultured, the more obvious the dendritic structure is. The cells of experiment group and the positive control group when cultured for 12 days had the most typical dendritic structure. The negative control group cells had no dendritic structure and became macrophages when cultured for 12 days. The experiment group cells cultured for 10 days showed typical dendritic morphology by SEM. The experiment group cells and the positive group cells cultured for 12 days significantly expressed high level of the phenotypes of DCs(CD1a, CD80, CD86 and CD83) by flowcytometry.And the difference exhibitied statistical significance when compared with the negative control group(P0.05).The mixed lymphocyte reaction showed that the DCs induced by APS trigerred proliferation of allogenic T cells obviously.Conclusion:Both APS and cytokine could induce the cord blood monocytes to differentiate into functional DCs committedly. DCs reduced by APS stimualate proliferation of the allogenic T cells obviously.