Objective:The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2(deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research.Methods:The huma...Objective:The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2(deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research.Methods:The human DBC2 gene was first subcloned into a shuttle plasmid pAdTrack-CMV.After recombining with pAdEasy-1 vector in BJ5183 cells,the new recombinant vector pAdEasy-DBC2-CMV was transfected into HEK-293 cells to produce adenovirus.The human bladder cancer cell line T24 was infected with DBC2-containing adenovirus particles.Both RNA and protein were collected from cells harvested at 72 h after infection.Real time quantitative PCR(qPCR) and Western blot were used to examine mRNA and protein levels.Fluorescence microscopy was utilized to observe the expression of reporter green fluorescence protein.Results:Electrophoresis showed there was a 2.2 kb size band produced from high fidelity PCR.Pac I digest of the final produced recombinant vector yielded band sizes of approximately 30 kb and 4.5 kb.After virus infection with the pAdEasy-DBC2-CMV vector,the T24 cell line was observed to highly express green fluorescence protein under a fluorescence microscope.qPCR and Western blot assay identified that the DBC2 gene was overexpressed at both the mRNA and protein levels in virus transfected cells.Conclusion:By using the pAdEasy adenovirus system,we successfully constructed an adenovirus that could highly overexpress the tumor suppressor DBC2 gene in a bladder cancer cell line.This viral construct would be widely used for our further research in gene functional assays and gene therapy in bladder cancer.展开更多
OBJECTIVE This study was conducted to explore the effect of Rg3 on inhibition of proliferation and induction of apoptosis in bladder cancer cells. METHODS The EJ bladder cancer cell line was treated with Rg3 at variou...OBJECTIVE This study was conducted to explore the effect of Rg3 on inhibition of proliferation and induction of apoptosis in bladder cancer cells. METHODS The EJ bladder cancer cell line was treated with Rg3 at various concentrations. Cell proliferation was measured by the MTT assay. Morphological changes in the cells were observed by fluorescent staining using Hoechst 33258. The cell cycle and apoptotic rate were analyzed by flow cytometry （FCM） and the expression of caspase-3 in cells was detected by immunocytochemistry. DNA ladder analysis was conducted by agarose gel electrophoresis. RESULTS Rg3 inhibited proliferation of EJ cells in a concentration-dependent manner, resulting in an IC50 for Rg3 at 48 h of 125.5 μg/ml. When treated with 150 μg/ml of Rg3 for 24 h and 48 h, the cells showed apoptotic morphological characteristics including condensed chromatin, nuclear fragmentation, apoptotic bodies and bright fluorescent granules as well as a higher caspase-3 expression. The FCM assay indicated that Rg3 altered the cell cycle and induced apoptosis of the EJ cells, when treated for 24 h and 48 h with 75 μg/ml of Rg3 as well as for 48 h with 150 μg/ml. The percentages of cells in the S phase and the GJM transition were increased, whereas the percentages of cells in the G0-G1 transition were decreased. The apoptotic rates were increased from （1.05±0.17）% in the control group cells to （8.41 ±0.98）%, （18.57±2.20）% and （33.98±1,64）% respectively. Significant changes in the DNA ladders, showed that the effects of Rg3 were displayed in a dose and time dependent manner. CONCLUSION The results suggest that Ginsenoside Rg3 exerts an inhibitory effect on proliferation of EJ cells by inducing apoptosis.展开更多
文摘Objective:The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2(deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research.Methods:The human DBC2 gene was first subcloned into a shuttle plasmid pAdTrack-CMV.After recombining with pAdEasy-1 vector in BJ5183 cells,the new recombinant vector pAdEasy-DBC2-CMV was transfected into HEK-293 cells to produce adenovirus.The human bladder cancer cell line T24 was infected with DBC2-containing adenovirus particles.Both RNA and protein were collected from cells harvested at 72 h after infection.Real time quantitative PCR(qPCR) and Western blot were used to examine mRNA and protein levels.Fluorescence microscopy was utilized to observe the expression of reporter green fluorescence protein.Results:Electrophoresis showed there was a 2.2 kb size band produced from high fidelity PCR.Pac I digest of the final produced recombinant vector yielded band sizes of approximately 30 kb and 4.5 kb.After virus infection with the pAdEasy-DBC2-CMV vector,the T24 cell line was observed to highly express green fluorescence protein under a fluorescence microscope.qPCR and Western blot assay identified that the DBC2 gene was overexpressed at both the mRNA and protein levels in virus transfected cells.Conclusion:By using the pAdEasy adenovirus system,we successfully constructed an adenovirus that could highly overexpress the tumor suppressor DBC2 gene in a bladder cancer cell line.This viral construct would be widely used for our further research in gene functional assays and gene therapy in bladder cancer.
文摘OBJECTIVE This study was conducted to explore the effect of Rg3 on inhibition of proliferation and induction of apoptosis in bladder cancer cells. METHODS The EJ bladder cancer cell line was treated with Rg3 at various concentrations. Cell proliferation was measured by the MTT assay. Morphological changes in the cells were observed by fluorescent staining using Hoechst 33258. The cell cycle and apoptotic rate were analyzed by flow cytometry （FCM） and the expression of caspase-3 in cells was detected by immunocytochemistry. DNA ladder analysis was conducted by agarose gel electrophoresis. RESULTS Rg3 inhibited proliferation of EJ cells in a concentration-dependent manner, resulting in an IC50 for Rg3 at 48 h of 125.5 μg/ml. When treated with 150 μg/ml of Rg3 for 24 h and 48 h, the cells showed apoptotic morphological characteristics including condensed chromatin, nuclear fragmentation, apoptotic bodies and bright fluorescent granules as well as a higher caspase-3 expression. The FCM assay indicated that Rg3 altered the cell cycle and induced apoptosis of the EJ cells, when treated for 24 h and 48 h with 75 μg/ml of Rg3 as well as for 48 h with 150 μg/ml. The percentages of cells in the S phase and the GJM transition were increased, whereas the percentages of cells in the G0-G1 transition were decreased. The apoptotic rates were increased from （1.05±0.17）% in the control group cells to （8.41 ±0.98）%, （18.57±2.20）% and （33.98±1,64）% respectively. Significant changes in the DNA ladders, showed that the effects of Rg3 were displayed in a dose and time dependent manner. CONCLUSION The results suggest that Ginsenoside Rg3 exerts an inhibitory effect on proliferation of EJ cells by inducing apoptosis.
