Human interleukin 17(IL17) is a newly found cytokine secreted by activated T lymphocytes. Many of its characteristics remain unknown. To provide a way for understanding its role in immunology and hematology further, w...Human interleukin 17(IL17) is a newly found cytokine secreted by activated T lymphocytes. Many of its characteristics remain unknown. To provide a way for understanding its role in immunology and hematology further, we constructed a recombinant retroviral vector pL17SN containing the human IL17 gene about 1.3kb with the coding region. The constructed retroviral vector packaged in CRIP cells could infect human primary fibroblasts, and could stimulate the primary fibroblasts secreting human GMCSF and IL6. The retroviral vector containing the human IL17 gene we constructed may present an efficient way to understand the biological functions of human IL17 and to investigate applications of IL17 in cancer gene therapy.展开更多
Both the antigen presenting ability and the cytotoxicity of macrophages can be enhanced by GMCSF gene transfer. In the present study, the therapeutic effect of intratumoral injection with GMCSF genemodified allogenic ...Both the antigen presenting ability and the cytotoxicity of macrophages can be enhanced by GMCSF gene transfer. In the present study, the therapeutic effect of intratumoral injection with GMCSF genemodified allogenic macrophages on tumorbearing mice observed. The peritoneal macrophages of C57BL/6 mice were transfected with GMCSF gene mediated by recombinant adenovirus and the subcutaneous CT26 colon adenocarcinomabearing BALB/c mice were treated by intratumoral injection of the above macrophages. The survival time of the tumorbearing mice were prolonged significantly and some tumor mass disappeared completely. The necroses of the tumor cells and massive infiltration of inflammatory cells were observed 6 days after treatment. 30 days after treatment, only the leftover of tumor cells and the inflammatory cells remained. The data indicated that introtumoral injection of GMCSF genemodified allogenic macrophages displayed more potent therapeutic effect on the preestablished tumorbearing mice.展开更多
In vitro human monocyteline U937 cells were inoculated with HSV1, which were persistently treated with 10 μg/ml LPS or/and 10 3 U/ml rhGMCSF as well as 10 3 U/ml rhIL3 since two days before inoculation. The effects o...In vitro human monocyteline U937 cells were inoculated with HSV1, which were persistently treated with 10 μg/ml LPS or/and 10 3 U/ml rhGMCSF as well as 10 3 U/ml rhIL3 since two days before inoculation. The effects of GMCSF and IL3 on the resistance of U937 cells to HSV1 were studied by microcytopathy assay for the infective titers of the cell culture supernatans(TCID 50 ). The results showed that at the 4 th day after inoculation the mean titers of GMCSF group and IL3 group were lower 44 and 21 fold than that of control group, respectively. The inhibition of HSV1 replication in LPSstimulated U937 cells induced by GMCSF or by IL3 was more significant. 2, 4, 6 and 8 days after inoculation, the mean titers of GMCSF+LPS group were lower 74, 162, 15 and 11 fold, and those of IL3+LPS group were lower 89, 18, 59 and 10 fold than that of only LPS treated group, respectively. These data indicate that GMCSF and IL3 could antagonise the enhancement activity of LPS for the virus replication in the cells and increase the resistance of U937 cells to HSV1.展开更多
Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retrovira...Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.Methods Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the “ping-pong' technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.Results The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6×105?cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7?μg/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4?ng*ml-1*106 cells-1*2 days-1.Conclusion The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.展开更多
文摘Human interleukin 17(IL17) is a newly found cytokine secreted by activated T lymphocytes. Many of its characteristics remain unknown. To provide a way for understanding its role in immunology and hematology further, we constructed a recombinant retroviral vector pL17SN containing the human IL17 gene about 1.3kb with the coding region. The constructed retroviral vector packaged in CRIP cells could infect human primary fibroblasts, and could stimulate the primary fibroblasts secreting human GMCSF and IL6. The retroviral vector containing the human IL17 gene we constructed may present an efficient way to understand the biological functions of human IL17 and to investigate applications of IL17 in cancer gene therapy.
文摘Both the antigen presenting ability and the cytotoxicity of macrophages can be enhanced by GMCSF gene transfer. In the present study, the therapeutic effect of intratumoral injection with GMCSF genemodified allogenic macrophages on tumorbearing mice observed. The peritoneal macrophages of C57BL/6 mice were transfected with GMCSF gene mediated by recombinant adenovirus and the subcutaneous CT26 colon adenocarcinomabearing BALB/c mice were treated by intratumoral injection of the above macrophages. The survival time of the tumorbearing mice were prolonged significantly and some tumor mass disappeared completely. The necroses of the tumor cells and massive infiltration of inflammatory cells were observed 6 days after treatment. 30 days after treatment, only the leftover of tumor cells and the inflammatory cells remained. The data indicated that introtumoral injection of GMCSF genemodified allogenic macrophages displayed more potent therapeutic effect on the preestablished tumorbearing mice.
文摘In vitro human monocyteline U937 cells were inoculated with HSV1, which were persistently treated with 10 μg/ml LPS or/and 10 3 U/ml rhGMCSF as well as 10 3 U/ml rhIL3 since two days before inoculation. The effects of GMCSF and IL3 on the resistance of U937 cells to HSV1 were studied by microcytopathy assay for the infective titers of the cell culture supernatans(TCID 50 ). The results showed that at the 4 th day after inoculation the mean titers of GMCSF group and IL3 group were lower 44 and 21 fold than that of control group, respectively. The inhibition of HSV1 replication in LPSstimulated U937 cells induced by GMCSF or by IL3 was more significant. 2, 4, 6 and 8 days after inoculation, the mean titers of GMCSF+LPS group were lower 74, 162, 15 and 11 fold, and those of IL3+LPS group were lower 89, 18, 59 and 10 fold than that of only LPS treated group, respectively. These data indicate that GMCSF and IL3 could antagonise the enhancement activity of LPS for the virus replication in the cells and increase the resistance of U937 cells to HSV1.
文摘Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.Methods Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the “ping-pong' technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.Results The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6×105?cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7?μg/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4?ng*ml-1*106 cells-1*2 days-1.Conclusion The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.
