An Experiment Study Of The Eukaryotic Expression And Inducing Apoptosis Effect Of VP3 Gene Combinated With Gemcitabine In Human Bladder Cancer Cell In Vitro

Objective:To explore the expression of chicken anemia virus(CAV)VP3 Gene in Human Bladder Cancer cell EJ cell.To observe apoptosis effect induced by chicken anemia virus(CAV)VP3 Gene,chicken anemia virus(CAV)VP3 Gene combination Gemcitabine in Human Bladder Cancer cell.Methods:By Lipofectamine^TM2000 mediated transfection in vitro,the recombinant eukaryotic expression vector PcDNA3-VP3 gene was transfectded into human bladder cancer cell lines EJ.Then,RT-PCR was performed to detect the expression condition of VP3 gene in vivo.Gemcitabine was divided into three gradients according to Gemcitabine's density:10^-7 mol/L,10^-8mol/L,10^-9mol/L.EJ cell also was divided into three groups:transfectded VP3 Gene,mixed Gemcitabine,VP3 Gene combination Gemcitabine.After that,for all these cells,MTT method was performed to detect the proliferation of cells;transmission electronmicroscope were used to determine the apoptotic morphological changes in cells; flow cytometry(FCM)using TUNAL method was operated to quantify apoptotic rates of cells.Results:The results of RT-PCR suggested that VP3 gene was expressed in EJ cells.The typical morphological changes of apoptosis such as cell shrinkage,chromatin condensation,nuclear fragmentation and apoptotic body were observed by both light and electron microscope.The proliferation of EJ cells which was detected by MTT method all decrease distinctively than that in control group(P＜0.01).After transfection for 24,48,and 72 hours,the apoptotic rates were(22.8±1.83%),(48.8±1.22%),(62.20±3.40%)in PcDNA3-VP3 group;(18.3±2.11%),(34.8±1.84%),(50.5±1.96%)in mixed Gemcitabine group;(35.2%±1.50%),(68.8±1.03%),(85.6±3.20%)in VP3 Gene combination Gemcitabine group.The apoptotic rates were all higher than that in control group(P＜0.01), and the apoptotic rates in VP3 Gene combination Gemcitabine group was higher than that in in PcDNA3-VP3 group.Conclusion:The expression of chicken anemia virus VP3 gene can induce high efficiency apoptosis in human bladder cancer cell lines EJ.At the same time,the results of our experimental data also suggest that Gemcitabine can enhance the efficiency of apoptosis which is induced by chicken anemia virus VP3 gene,which will provide a foundation for the future work such as to manufacture protein and function researching of this fusion protein.