Objective：To study the immunologic function of dendritic cells（DCs） cultured in two kinds of hepatoma cell line＇s supernatant and the enhancing effects of carboxymethylpachymaran（CMP） on DCs.Methods：DCs were ha...Objective：To study the immunologic function of dendritic cells（DCs） cultured in two kinds of hepatoma cell line＇s supernatant and the enhancing effects of carboxymethylpachymaran（CMP） on DCs.Methods：DCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor （GM-CSF） and interleukin（IL）-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines（HepG2 and HepG2.2.15） were collected for condition medium（CM） according to a volume ratio of supernatant to incomplete RPMI-1640 medium,which was 3：1.CMP was dissolved in incomplete RPMI-1640 medium.Experimental groups were divided according to the culture medium,either CM or with CMP in it.DCs subsets CD83,CD86,CD1a,and d-related human leukocyte antigens（HLA-DR） were analyzed by flow cytometry.The proliferation ability of allogeneic T cells in mixed lymphocyte reaction（MLR） stimulated by DCs was examined by 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide（MTT） analysis.IL-12p70,interferon-γ（IFN-γ）,and nuclear factor k B（NF- k B） were detected by enzyme-linked immunosorbent assay analysis.Results：The proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group（P0.01）.Compared with the normal group,groups treated with CMP showed a higher expression level of DCs subsets,lymphocyte reproductive activity,as well as IL-12 and IFN-γsecretion levels.Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γsecretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group（P0.05）.Compared with the normal group,the expression level of NF-k B in DCs nuclear was higher in CMP groups but lower in two CM groups（P0.05）.After CMP was added,the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added（P0.05）.However,there was no significant difference between the two CM groups（P0.05）.Conclusions：Two kinds of hepatoma cell line＇s supernatant can inhibit the immunologic function of DCs.This suppressive effect may be related to the inhibition of NF-κB/Rel pathway.CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.展开更多
基金Supported by the National Natural Science Foundation of China (No.30973693)the Research Fund for the Doctoral Program of Higher Education of China(No.20104401110003)
文摘Objective：To study the immunologic function of dendritic cells（DCs） cultured in two kinds of hepatoma cell line＇s supernatant and the enhancing effects of carboxymethylpachymaran（CMP） on DCs.Methods：DCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor （GM-CSF） and interleukin（IL）-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines（HepG2 and HepG2.2.15） were collected for condition medium（CM） according to a volume ratio of supernatant to incomplete RPMI-1640 medium,which was 3：1.CMP was dissolved in incomplete RPMI-1640 medium.Experimental groups were divided according to the culture medium,either CM or with CMP in it.DCs subsets CD83,CD86,CD1a,and d-related human leukocyte antigens（HLA-DR） were analyzed by flow cytometry.The proliferation ability of allogeneic T cells in mixed lymphocyte reaction（MLR） stimulated by DCs was examined by 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide（MTT） analysis.IL-12p70,interferon-γ（IFN-γ）,and nuclear factor k B（NF- k B） were detected by enzyme-linked immunosorbent assay analysis.Results：The proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group（P0.01）.Compared with the normal group,groups treated with CMP showed a higher expression level of DCs subsets,lymphocyte reproductive activity,as well as IL-12 and IFN-γsecretion levels.Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γsecretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group（P0.05）.Compared with the normal group,the expression level of NF-k B in DCs nuclear was higher in CMP groups but lower in two CM groups（P0.05）.After CMP was added,the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added（P0.05）.However,there was no significant difference between the two CM groups（P0.05）.Conclusions：Two kinds of hepatoma cell line＇s supernatant can inhibit the immunologic function of DCs.This suppressive effect may be related to the inhibition of NF-κB/Rel pathway.CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.
