To investigate the protective effect of epigallocatechin gallate （EGCG） on the immune function of dendritic cells （DCs） after ultraviolet B irradiation （UVB） and its underlying mechanisms, the monocytes were iso...To investigate the protective effect of epigallocatechin gallate （EGCG） on the immune function of dendritic cells （DCs） after ultraviolet B irradiation （UVB） and its underlying mechanisms, the monocytes were isolated from peripheral blood and cultivated into DCs with cytokines, such as GM-CSF and IL-4. DCs were harvested after cultivation for 7 d and subjected to irradiation with different dosages of UVB. Then, 200 μg/ml of EGCG were added in certain groups immediately after irradiation. DCs simply treated with UVB or treated with both UVB and EGCG were co-cultured with lymphocytes, and MTT assay was used to detect the ability of DCs to stimulate proliferation of lymphocytes. Surface markers CDS0, CD86, HLA-DR and CD40 were detected by flow cytometry, and the levels of IL-10 and IL-12 secreted from DCs 2d h after cultivation were measured by ELISA. It was demonstrated that UVB irradiation could inhibit the ability of DCs to stimulate the proliferation of lymphocytes and surface expressions of CDS0, CD86, HLA-DR and CD40 on DCs in a dose-dependent manner. The inhibition rate of DCs was improved to some extent after treatment with 200μg/ml of EGCG. When the concentra- tion of EGCG exceeded 100 μg/ml, the enhancing effect of EGCG on the expression of the co-stimulating molecules on DCs could be demonstrated in a dose-dependent manner. UVB showed no significant influence on the secretion of IL-10 and IL-12 from DCs, while EGCG could down-regulate the secretion level of IL-12 and up-regulate that of IL-10. It is concluded that EGCG can antagonize the inhibitory effect on DCs induced by UVB irradiation. This function has some relationship with its protecting effect of the expression of the co-stimulating molecule on the surface of DCs and the secretion level of IL-10 and IL-12.展开更多
Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal f...Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell re- ceptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, result- ing in the phosphorylation of Syk and Erkl/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.展开更多
文摘To investigate the protective effect of epigallocatechin gallate （EGCG） on the immune function of dendritic cells （DCs） after ultraviolet B irradiation （UVB） and its underlying mechanisms, the monocytes were isolated from peripheral blood and cultivated into DCs with cytokines, such as GM-CSF and IL-4. DCs were harvested after cultivation for 7 d and subjected to irradiation with different dosages of UVB. Then, 200 μg/ml of EGCG were added in certain groups immediately after irradiation. DCs simply treated with UVB or treated with both UVB and EGCG were co-cultured with lymphocytes, and MTT assay was used to detect the ability of DCs to stimulate proliferation of lymphocytes. Surface markers CDS0, CD86, HLA-DR and CD40 were detected by flow cytometry, and the levels of IL-10 and IL-12 secreted from DCs 2d h after cultivation were measured by ELISA. It was demonstrated that UVB irradiation could inhibit the ability of DCs to stimulate the proliferation of lymphocytes and surface expressions of CDS0, CD86, HLA-DR and CD40 on DCs in a dose-dependent manner. The inhibition rate of DCs was improved to some extent after treatment with 200μg/ml of EGCG. When the concentra- tion of EGCG exceeded 100 μg/ml, the enhancing effect of EGCG on the expression of the co-stimulating molecules on DCs could be demonstrated in a dose-dependent manner. UVB showed no significant influence on the secretion of IL-10 and IL-12 from DCs, while EGCG could down-regulate the secretion level of IL-12 and up-regulate that of IL-10. It is concluded that EGCG can antagonize the inhibitory effect on DCs induced by UVB irradiation. This function has some relationship with its protecting effect of the expression of the co-stimulating molecule on the surface of DCs and the secretion level of IL-10 and IL-12.
基金supported by the National Basic Research Program of China(2011CB965203)to Yue Huang
文摘Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell re- ceptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, result- ing in the phosphorylation of Syk and Erkl/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.
