| Objective With regard to the low expression extremely of coxsackie adenovirus receptor(CAR) in high grade/stage bladder cancer cell, adenovirus serotype 5(Ad5) is limited to develop a high efficient capacity of transduction and oncolysis. In this study,we aimed to construct a novel of CAR-independent oncolytic adenovirus Ad5/F11-PSCAE-UPII-E1A(Ad5/F11) through altering the tropism of Ad5, namely combining the fiber region from Ad11 with the base domain from Ad5 while inserting into the uroplakin II(UPII) promotor and prostate stem cell antigen enhancer(PSCAE) before the E1 A gene for a dual specific regulation. The bladder cancer cell lines EJ and T24 were perfomed to observe the changes in the cellular morphology and proliferation and figure out the optimal multiplicity of infection(MOI) after administration with Ad5/F11. And we then investigated the influence of Ad5 and Ad5/F11 on transduction efficacy and therapeutic effect of EJ and T24. All of these data were expected to provide a new approach and establish an experimental basis for the following gene therapy of bladder cancer.Methods The shuttle plasmid Rp-PSCAE-UPII-E1 A which was preserved in our lab and the modified skeleton construction Ad5/F11 were transfected into competent E. coli BJ5183 for homologous recombination. The positive bacterial strain containing recombinant plasmid were screened by kanamycin.The extracted recombinant plasmid was regared as template and amplified the target gene UPII、PSCAE and E1 A by polymerase chain reaction(PCR), digested with PacI and BsrGI as well. After agarose gel electrophoresis, the correct recombinant adenovirus plasmid was identified. The purified adenovirus plasmid was used to transfected into HEK 293 for packaging in a vary of proportional concentration. The position and size of target genes were detected by PCR via extracting the genome of adenovirus. The sufficient adenovirus were purified using reagent and measured the titer accoring to the TCID50.Compared with HEK293, furthermore, the relative mRNA expression of CD46 in EJ and T24 were analysized by means of real-time PCR. Conducting to infect EJ and T24 with different gradient MOI of Ad5/F11, We examined the anti-tumor effect in CCK8 assay. Selecting optimal MOI of Ad5/F11, subsequently, we compared the therapeutic results and cells viability of EJ and T24 with Ad5 in 8 days.Result The recombinant adenovirus, with the high titer of 1×1010PFU/ml, was successfully reconstructed. RT-PCR assay demonstrated that the mRNA expression of CD46 is significantly higher in EJ and T24 than that in HEK293. In our CCK8 assay,the sharpest decline of cell viability was obtained as EJ and T24 were interfere with Ad5/F11 range from 20 to 40 MOI. Infecting EJ and T24 with respectively optimal MOI of Ad5/F11 and Ad5 in a observation period of 8 days, the cell viability of EJ and T24 in Ad5/F11 group were 19.6±3.48% and 51.8±2.48%, unfortunately, that were 33.38±2.54% in EJ and 80.27±2.18% in T24 in Ad5 group.Conclusion The recombinant adenovirus Ad5/F11-PSCAE-UPII-E1 A which was characterized by replicating selectively in bladder cancer with high titer was obtained. With the strong therapeutic effect in EJ and T24, Ad5/F11 was supposed to be superior to Ad5. Therefore, improving the transduction efficacy of adenovirus is responsible for reinforcing oncolysis for tumor. These data established a preliminary experimential basis for the preferable chioce in the management of bladder cancer. |
PDF Full Text Request