Background Gene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-in...Background Gene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-inducible dual-gene co-expression vector pEgr-interferon(IFN)-γ- endostatin and studied the anti-tumor effect of pEgr-IFN-γ-endostatin gene-radiotherapy in mice bearing Lewis lung carcinoma and its mechanism.Methods Gene recombinant technique was used to construct dual-gene co-expression plasmid pEgr-IFN-γ-endostatin, and single-gene expression plasmid pEgr-IFN-γ and pEgr-endostatin. The plasmids packed by liposome were injected locally into the tumors of the mice, and the tumors were irradiated with 5 Gy X-ray 36 hours later. The tumor growth rate at different time and mean survival period of the mice were observed. Cytotoxic activity of splenic cytotoxic T-lymphocyte (CTL), natural killer (NK) cell and tumor necrosis factor (TNF)-α secretion activity of peritoneal macrophages of the mice in various groups were evaluated 15 days after irradiation. The intratumor micro-vessel density was evaluated by immunohistochemical staining 10 days after irradiation.Results The tumor growth rate of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy group and single-gene-radiotherapy group at different time after gene-radiotherapy, and the mean survival period of which was longer. Cytotoxic activity of splenic CTL, NK and TNF-α secretion activity of peritoneal macrophages of the mice in dual-gene-radiotherapy group were significantly higher than those in control group, 5 Gy X-ray irradiation group and pEgr-endostatin gene-radiotherapy group 15 days after irradiation. The intratumor micro-vessel density of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy X-ray irradiation group and pEgr-IFN-γgene-radiotherapy group. Conclusion The anti-tumor effect of dual-gene-radiotherapy was significantly better than that of s展开更多
目的 :构建携带人肿瘤坏死因子相关凋亡诱导配体 (h TRAIL )基因的辐射诱导表达载体p Egr- h TRAIL ,并研究其在人肺腺癌细胞株 A 5 4 9中的表达及促凋亡作用。方法 :采用常规分子生物学方法构建表达质粒 p Egr- h TRAIL ,体外通过脂质...目的 :构建携带人肿瘤坏死因子相关凋亡诱导配体 (h TRAIL )基因的辐射诱导表达载体p Egr- h TRAIL ,并研究其在人肺腺癌细胞株 A 5 4 9中的表达及促凋亡作用。方法 :采用常规分子生物学方法构建表达质粒 p Egr- h TRAIL ,体外通过脂质体转染 A5 4 9细胞 ,经 G4 18筛选稳定转染细胞 A 5 4 9- sh TRAIL ,利用逆转录聚合酶链反应 (RT- PCR)法检测目的基因的表达 ,采用 Annexin- V- FITC凋亡检测试剂盒检测细胞早期凋亡。结果 :经限制性内切酶酶切鉴定 p Egr- h TRAIL表达质粒构建正确 ;稳定转染细胞 A 5 4 9- sh TRAIL的 h TRAILm RNA表达量明显增加 ;稳定转染细胞 A 5 4 9- sh TRAIL的凋亡细胞百分数明显增加 ,是 A5 4 9细胞的 1.8倍(P<0 .0 5 )。结论 :成功构建 p Egr- h TRAIL表达质粒 ,体外稳定转染细胞 A5 4 9- sh TRAIL具有明显诱导凋亡作用。展开更多
AIM: To construct the recombinant pEgr-P16 plasmid for the investigation of its expression properties in esophageal squamous cell carcinoma induced by ionizing irradiation and the feasibility of gene-radiotherapy for ...AIM: To construct the recombinant pEgr-P16 plasmid for the investigation of its expression properties in esophageal squamous cell carcinoma induced by ionizing irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma.METHODS: The recombinant pEgr-P16 plasmid was constructed and transfected into EC9706 cells with lipofectamine. Western blot, quantitative RT-PCR and flow cytometry were performed to study the expression of pEgr-P16 in EC9706 cells and the biological characteristics of EC9706 cell line after transfection induced by ionizing irradiation.RESULTS: The eukaryotic expression vector pEgr-P16 was successfully constructed and transfected into EC9706 cells.The expression of P16 was significantly increased in the transfected cells after irradiation while the transfected cells were not induced by ionizing irradiation. The induction of apoptosis in transfection plus irradiation group was higher than that in plasmid alone or irradiation alone.CONCLUSION: The combination of pEgr-P16 and irradiation could significantly enhance the P16 expression property and markedly induce apoptosis in EC9706 cells. These results may lay an important experimental basis for gene radiotherapy for esophageal carcinoma.展开更多
目的 :克隆小鼠内皮抑素 (m Endostatin)编码区 c DNA序列并构建含 Egr- 1启动子的 IFNγ和 m Endo-statin双基因表达载体。方法 :利用逆转录多聚酶链反应法 (RT- PCR) ,以小鼠肝细胞 m RNA为模板 ,扩增获得全长 m Endostatin,与 p MD1...目的 :克隆小鼠内皮抑素 (m Endostatin)编码区 c DNA序列并构建含 Egr- 1启动子的 IFNγ和 m Endo-statin双基因表达载体。方法 :利用逆转录多聚酶链反应法 (RT- PCR) ,以小鼠肝细胞 m RNA为模板 ,扩增获得全长 m Endostatin,与 p MD1 8T载体连接作全自动测序 ,并利用基因重组技术构建含 Egr- 1启动子的 IFNγ和m Endostatin双基因表达质粒。结果 :经测序证实获得的 m Endostatin序列与文献报道完全一致 ,并构建了含 Egr-1启动子的 IFNγ和 m Endostatin双基因表达质粒 p Egr- IFNγ- m Endostatin。结论 :利用 RT- PCR法成功克隆了m Endostatin的 c DNA序列 ,构建了 p Egr- IFNγ- m Endostatin重组双基因表达质粒。展开更多
文摘Background Gene-radiotherapy, the combination of gene therapy and radiation therapy, is a new paradigm for cancer treatment. To enhance anti-tumor effect of gene-radiotherapy, in this study we construct a radiation-inducible dual-gene co-expression vector pEgr-interferon(IFN)-γ- endostatin and studied the anti-tumor effect of pEgr-IFN-γ-endostatin gene-radiotherapy in mice bearing Lewis lung carcinoma and its mechanism.Methods Gene recombinant technique was used to construct dual-gene co-expression plasmid pEgr-IFN-γ-endostatin, and single-gene expression plasmid pEgr-IFN-γ and pEgr-endostatin. The plasmids packed by liposome were injected locally into the tumors of the mice, and the tumors were irradiated with 5 Gy X-ray 36 hours later. The tumor growth rate at different time and mean survival period of the mice were observed. Cytotoxic activity of splenic cytotoxic T-lymphocyte (CTL), natural killer (NK) cell and tumor necrosis factor (TNF)-α secretion activity of peritoneal macrophages of the mice in various groups were evaluated 15 days after irradiation. The intratumor micro-vessel density was evaluated by immunohistochemical staining 10 days after irradiation.Results The tumor growth rate of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy group and single-gene-radiotherapy group at different time after gene-radiotherapy, and the mean survival period of which was longer. Cytotoxic activity of splenic CTL, NK and TNF-α secretion activity of peritoneal macrophages of the mice in dual-gene-radiotherapy group were significantly higher than those in control group, 5 Gy X-ray irradiation group and pEgr-endostatin gene-radiotherapy group 15 days after irradiation. The intratumor micro-vessel density of the mice in dual-gene-radiotherapy group was significantly lower than those in control group, 5 Gy X-ray irradiation group and pEgr-IFN-γgene-radiotherapy group. Conclusion The anti-tumor effect of dual-gene-radiotherapy was significantly better than that of s
文摘目的 :构建携带人肿瘤坏死因子相关凋亡诱导配体 (h TRAIL )基因的辐射诱导表达载体p Egr- h TRAIL ,并研究其在人肺腺癌细胞株 A 5 4 9中的表达及促凋亡作用。方法 :采用常规分子生物学方法构建表达质粒 p Egr- h TRAIL ,体外通过脂质体转染 A5 4 9细胞 ,经 G4 18筛选稳定转染细胞 A 5 4 9- sh TRAIL ,利用逆转录聚合酶链反应 (RT- PCR)法检测目的基因的表达 ,采用 Annexin- V- FITC凋亡检测试剂盒检测细胞早期凋亡。结果 :经限制性内切酶酶切鉴定 p Egr- h TRAIL表达质粒构建正确 ;稳定转染细胞 A 5 4 9- sh TRAIL的 h TRAILm RNA表达量明显增加 ;稳定转染细胞 A 5 4 9- sh TRAIL的凋亡细胞百分数明显增加 ,是 A5 4 9细胞的 1.8倍(P<0 .0 5 )。结论 :成功构建 p Egr- h TRAIL表达质粒 ,体外稳定转染细胞 A5 4 9- sh TRAIL具有明显诱导凋亡作用。
基金the National Natural Science Foundation of China,No.30210103904 the Science and Technology Program of Gnangdong Province,No.2003C30304
文摘AIM: To construct the recombinant pEgr-P16 plasmid for the investigation of its expression properties in esophageal squamous cell carcinoma induced by ionizing irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma.METHODS: The recombinant pEgr-P16 plasmid was constructed and transfected into EC9706 cells with lipofectamine. Western blot, quantitative RT-PCR and flow cytometry were performed to study the expression of pEgr-P16 in EC9706 cells and the biological characteristics of EC9706 cell line after transfection induced by ionizing irradiation.RESULTS: The eukaryotic expression vector pEgr-P16 was successfully constructed and transfected into EC9706 cells.The expression of P16 was significantly increased in the transfected cells after irradiation while the transfected cells were not induced by ionizing irradiation. The induction of apoptosis in transfection plus irradiation group was higher than that in plasmid alone or irradiation alone.CONCLUSION: The combination of pEgr-P16 and irradiation could significantly enhance the P16 expression property and markedly induce apoptosis in EC9706 cells. These results may lay an important experimental basis for gene radiotherapy for esophageal carcinoma.
文摘目的 :克隆小鼠内皮抑素 (m Endostatin)编码区 c DNA序列并构建含 Egr- 1启动子的 IFNγ和 m Endo-statin双基因表达载体。方法 :利用逆转录多聚酶链反应法 (RT- PCR) ,以小鼠肝细胞 m RNA为模板 ,扩增获得全长 m Endostatin,与 p MD1 8T载体连接作全自动测序 ,并利用基因重组技术构建含 Egr- 1启动子的 IFNγ和m Endostatin双基因表达质粒。结果 :经测序证实获得的 m Endostatin序列与文献报道完全一致 ,并构建了含 Egr-1启动子的 IFNγ和 m Endostatin双基因表达质粒 p Egr- IFNγ- m Endostatin。结论 :利用 RT- PCR法成功克隆了m Endostatin的 c DNA序列 ,构建了 p Egr- IFNγ- m Endostatin重组双基因表达质粒。
