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展开更多
Objective： To observe the anti-virus effects of andrographolide （AD） on the retinoic acid-inducible gene-I （RIG-I）-Iike receptors （RLRs） signaling pathway when immunological cells were infected with HIN1. Meth...Objective： To observe the anti-virus effects of andrographolide （AD） on the retinoic acid-inducible gene-I （RIG-I）-Iike receptors （RLRs） signaling pathway when immunological cells were infected with HIN1. Methods： Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line （16HBE）. The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium （MTT） assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma （IFN- ~, ） and interleukin-4 （IL-4） enzyme-linked immunosorbent assay （ELISA） detection while immunological cells for real-time polymerase chain reaction （RT-PCR）. Results： The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-13 promoter stimulator-1 （IPS-1）, interferon regulatory factor （IRF）-7, IRF-3 and nuclear transcription factor K B （NF- K B） mRNA levels increased significantly （P〈0.05）. When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF- K B decreased significantly in each group with significant statistic differences （P〈0.05）. Conclusions： The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling 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
基金Supported by the National Natural Science Foundation of China(No.81273616 and 30973693)the Doctoral Fund of the Ministryof Education 2010(No.20104401110003)the Natural Science Foundation of Guangdong Province(No.S2013010013434)
文摘Objective： To observe the anti-virus effects of andrographolide （AD） on the retinoic acid-inducible gene-I （RIG-I）-Iike receptors （RLRs） signaling pathway when immunological cells were infected with HIN1. Methods： Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line （16HBE）. The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium （MTT） assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma （IFN- ~, ） and interleukin-4 （IL-4） enzyme-linked immunosorbent assay （ELISA） detection while immunological cells for real-time polymerase chain reaction （RT-PCR）. Results： The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-13 promoter stimulator-1 （IPS-1）, interferon regulatory factor （IRF）-7, IRF-3 and nuclear transcription factor K B （NF- K B） mRNA levels increased significantly （P〈0.05）. When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF- K B decreased significantly in each group with significant statistic differences （P〈0.05）. Conclusions： The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.
