Objective: To investigate the feasibility of temporally and spatially restricted Kringle5 expression induced by radiation, as well as the dual effect of radiotherapy and antiangiogenic therapy in lung adenocarcinoma i...Objective: To investigate the feasibility of temporally and spatially restricted Kringle5 expression induced by radiation, as well as the dual effect of radiotherapy and antiangiogenic therapy in lung adenocarcinoma in vitro. Methods: We first constructed recombinant baculovirus vectors containing Egr1 promoter and human plasminogen Kringle5 gene (rhK5), then transfected them into lung adenocarcinoma cells (A549). Transfect efficiency of the baculovirus for gene transfer in A549 cells and the activity of Egr1 promoter induced by X-radiation were detected by fluorescence microscopy. The rhK5 mRNA transcription and rhK5 protein expression were detected by Real-time PCR and Western blot assay, respectively. The apoptosis asssay of human umbilical veins endothelial cells (HUVEC) was analyzed by flow cytometry. Results: The recombinant baculovirus were successfully transfected into A549 and HUVEC cells. As for the temporal regulation, the rhK5 mRNA transcription and rhK5 protein expression were elevated with the irradiation time significantly. And the HUVEC apoptotic percentage increased in relation to the irradiation time as well. As for the spatial regulation, rhK5 mRNA transcription level of A549 cell lines transfected with recombinant baculovirus Egr1-K5 was significantly higher than that of control groups after the same dose of X-radiation. When we analyzed the dose and frequency of X-radiation, no difference was observed among each dose after continuously three-times of irradiation. Conclusion: Baculovirus-mediated Egr1-K5 can be used in gene radiotherapy for its temporary and spatial controllable rhK5 expression by X-radiation and the consequent HUVEC apoptosis in vitro study. And low dose and more times of irradiation might be more effective. It would provide a promising way for the tumor treatment.展开更多
文摘Objective: To investigate the feasibility of temporally and spatially restricted Kringle5 expression induced by radiation, as well as the dual effect of radiotherapy and antiangiogenic therapy in lung adenocarcinoma in vitro. Methods: We first constructed recombinant baculovirus vectors containing Egr1 promoter and human plasminogen Kringle5 gene (rhK5), then transfected them into lung adenocarcinoma cells (A549). Transfect efficiency of the baculovirus for gene transfer in A549 cells and the activity of Egr1 promoter induced by X-radiation were detected by fluorescence microscopy. The rhK5 mRNA transcription and rhK5 protein expression were detected by Real-time PCR and Western blot assay, respectively. The apoptosis asssay of human umbilical veins endothelial cells (HUVEC) was analyzed by flow cytometry. Results: The recombinant baculovirus were successfully transfected into A549 and HUVEC cells. As for the temporal regulation, the rhK5 mRNA transcription and rhK5 protein expression were elevated with the irradiation time significantly. And the HUVEC apoptotic percentage increased in relation to the irradiation time as well. As for the spatial regulation, rhK5 mRNA transcription level of A549 cell lines transfected with recombinant baculovirus Egr1-K5 was significantly higher than that of control groups after the same dose of X-radiation. When we analyzed the dose and frequency of X-radiation, no difference was observed among each dose after continuously three-times of irradiation. Conclusion: Baculovirus-mediated Egr1-K5 can be used in gene radiotherapy for its temporary and spatial controllable rhK5 expression by X-radiation and the consequent HUVEC apoptosis in vitro study. And low dose and more times of irradiation might be more effective. It would provide a promising way for the tumor treatment.
