Effect Of Adenovirus-mediated Human Inducible Nitric Oxide Synthase Gene In TCC Cells

Bladder cancer is the most common tumor of urinary tract. With the improvement of living conditions and changes in the environment, incidence of the disease was increased year by year. More than 90%of bladder cancer are transitional cell carcinoma, including superficial bladder cancer about 70%. Treatment of superficial bladder cancer preferred transurethral resection of bladder tumor. For the prevention of high recurrence rate of TURBT postoperative intravesical perfusion therapy is currently the most effective treatment. The intravesical perfusion of HCPT, pirarubicin, BCG, etc are the most effective and commonly used methods. After treatment of perfusion, recurrence rate of TURBT of superficial TCC may be reduced by about 20%, but the recurrence rate is still 30-50%. And no matter what prevention and treatment measures,about 10-15% incidence of myometrial invasion to the bladder will not change. Postoperative adjuvant treatment of bladder cancer is currently considered most important.In recent years, studies have shown that NO is one of the regulatory signals that necessary for apoptosis of tumor cells.NO play a two-way role in tumor occurrence and development. Experiments confirmed that The concentration of NO in the mediated cytotoxicity or apoptosis is more than which in promoting angiogenesis 10-100-fold. It means the bi-directional role of NO dependenting on concentration. Appropriate low concentration will promote tumor growth and high concentration inhibit tumor production and promote the apoptosis. Therefore, stimulation the release of high concentrations of NO in tumor cells may be a new way to treat the cancer. A variety of ways can achieve high concentrations of NO in cells, such as transfected iNOS gene in cancer cells and direct complement of exogenous NO and so on. But the former is superior to the direct complement of exogenous NO. Therefore, endogenous and exogenous NOS will be a promising therapeutic agent or adjuvant chemotherapy.Compared with other areas, the study of biological effects about nitric oxide in bladder transitional cell carcinoma still has some gaps. No study has been reported at home and abroad that iNOS be applied to bladder transitional cell carcinoma.There are a variety of ways about synthesis and secretion of to high concentrations of NO. Some literature report using NO donor, such as nitro-vasodilators, as a simulation of high concentrations of NO in vivo, but there are some defects, such as poor drug resistance and side effects. Also some program have been reported,which through the activation of lymphocytes directly regulating the host immune response. The treatment of this area is mainly dependent on IFN-a, P, y, and IL-2, because of lacking specificity, toxic and other factors,which have never been widely used. Therefore, using NO donor drugs or cytokines producing NO,there are many limitations. Gene therapy refers to the use of genetic engineering method to transfer a specific foreign genes into animal or human tissue cells, making the integration, expression, in order to achieve therapeutic goals. The complexity of tumor to make the right choice for gene transfer methods and gene therapy strategy to become the key of gene therapy. This study attempts by gene transfection method to transfer exogenous iNOS into T24 bladder transitional cell cancer, increasing intracellular iNOS gene expression. In the role of iNOS, the concentration of NO in cells increased.It means the establishment of a cell NO donor, which can release of a large number of NO and interfere with cell growth cycle and induce cell apoptosis.With adenovirus-mediated gene transfer technology in cancer gene therapy is more common, the target gene can be integrated into the virus genome and then infected with host cells. Adenovirus has characteristics of addicting to the bladder cells,and the bladder is a semi-open organ, which makes application of adenovirus vectors for gene therapy for bladder cancer becoming possible. Gene therapy combined with existing treatments for against bladder cancer recurrence become the new ideas and ways. At home and abroad there is no study about applying iNOS to the bladder transitional cell carcinoma. We selected this study on the basis of past research and we hope appliying iNOS gene therapy to the bladder transitional cell carcinoma can be effective for clinical treatment of bladder transitional cell carcinoma. This issue Objectives:To construct the recombinant adenovirus vector coding inducible nitric oxide synthase gene and get the recombinant adenovirus of rAd-iNOS which was enrolled for researching the effects on T24 cell. Basic on previous work, we opened reading frame for iNOS gene and subcloned into adenovirus shuttle vector pYr-adshuttle-1, which was then reconstructed with adenovirus vector pAd/BL-DEST in vitro. The recombinant adenovirus vector was successfully constructed after we transfect pAd/BL-DEST to HEK293 cell.Methods:Sequencing of the plasmid template was used to verify pReceive-M29-iNOS. The band of iNOS fragments and pYr-adshuttle-1 were isolated from pReceive-M29-iNOS and adenovirus shuttle vector pYr-adshuttle-1 by restriction enzyme digestion and verified by agarose gel electrophoresis, and then was ligated and transferred to competent E. coli DH5a which was shaked and amplified for isolating recombinant plasmid, pYr-adshuttle-1-iNOS. This plasmid was verified by restriction enzyme digestion and DNA sequencing. Subcoloned iNOS expression cassette to adenovirus express vector pAd/BL-DEST by LR reaction in vitro and digestion on recombinant adenovirus vector pAd-iNOS with enzyme PacI. Transfect the cell HEK293 with linearizated recombinant adenovirus vector and packaged to reconstructed adenovirus rAd-iNOS which was verified by PCR and viral titer was determined by the viral particle (VP) method.Results:DNA sequence of plasmid template pReceive-M29-iNOS is consistent.with the expected results, the accuracy of compare the ORF of iNOS with the sequence published by NCBI is up to 100%. Restriction enzyme digestion and sequencing demonstrated successful construction of adenovirus shuttle vector pYr-adshuttle-1-iNOS and adenovirus vector pAd-iNOS. The recombinant adenovirus rAd-iNOS was successfully packaged and the viral titer was 5.8×108 PFU/ml.Conclusions:We had successfully constructed recombinant adenovirus shuttle vector pYr-adshuttle-1-iNOS and recombinant adenovirus vector pAd-iNOS. At last, recombinant adenovirus rAd-iNOS was generated by packaging recombinant adenovirus vector with cell HEK 293.Objectives:Study the effects of recombinant adenovirus rAd-iNOS on growth, proliferation and apoptosis of T24 cell.Methods:T24 cell had been cultured for 24 hours and Optimal multiplicity of infection was determined with recombinant adenovirus which express green fluorescent protein(GFP). Cells T24 during exponential phase of growth were enrolled into 3 groups 24 hours before infected:Group 1, Infected with recombinant adenovirus rAd-iNOS; Group 2, Infected with adenovirus rAd-NC as control; Group 3, blank. Added 100Mol recombinant adenovirus rAd-iNOS or adenovirus rAd-NC when the cell density reach to 60%-70%, cultured at 37℃,5%CO2 for 4 hours. Replaced with fresh complete medium and cultured for another 48 hours. The cells were harvested by digestion with trypsin. The mRNA expression of iNOS, p53 on T24 cells were tested by RT-PCR. The differentiations of protein expression of iNOS and p53 on T24 cells was detected by Western-blotting. NO test kit was used to demonstrate the secretion of NO. Cell apoptosis was monitored under the fluorescence microcope. Cell apoptosis in each group were detected with flow cytometry.Results:Cultured with recombinant adenovirus rAd-iNOS for 48 hours, mRNA of iNOS and p53 gene on T24 cells in group cultured with rAd-iNOS increased significantly compare to rAd-NC and blank group. Protein expression of iNOS,p53 on T24 cells cultured with rAd-iNOS was higher than group of T24 cells cultured with rAd-NOS and blank group. NO secretion was significantly increased in T24 cells treated with recombinant adenovirus rAd-iNOS(p<0.05). Under the fluorescent microscope, T24 cells cultured with recombinant adenovirus rAd-iNOS for 24 hours showed variation of growth and reduced malignant phenotype. Flow cytometry demonstrated that the apoptosis of T24 cells cultured with recombinant adenovirus rAd-iNOS is significantly higher than group control and blank(p<0.05).Conclusions:Transfected with recombinant adenovirus rAd-iNOS, T24 cell of bladder transitional carcinoma expressed increased iNOS protein and induced NO secretion. Expression of p53 protein was also increased significantly and promoted the apoptosis of T24 cells. This study found a noval path of gene therapy for bladder cancer and laid a solid basis for further research.Objectives:To explore the cell-killing effects of recombinant adenovirus rAd-iNOS on human transitional bladder carcinoma cell, we treated T24 cells with rAd-iNOS combined with low dosage chemotherapy.Methods:Culture T24 cells in flate bottomed 96-wells in culture medium, three wells for each group. Incubate the plate for 6-8 hours and transfect the genotype group with rAd-iNOS, T24 cells without transfect as control. Reference to previous data, the final concentration of HCPT and THP is harmless to cells. Add rAd-iNOS,rAd-iNOS+HCPT (THP) into each group, T24 cells without treatment as control. Morphological change of the cells was observed under the microscope. Survive ratio of the cells treated for 24,48, and 72 hours was analysized by MTT.Results:Compared with group transfect with rAd-iNOS, the killing efficacy of group rAd-iNOS+HCPT on tranfect cells increased significantly. With the increased concentration of HCPT and the time extension, the efficiency of cell killing increased. The efficiency of cell killing of group rAd-iNOS+HCPT on T24 cells increased comparing to group rAd-iNOS and blank. There are difference of cell killing efficiency among groups with different concentration of HCPT.Conclusions:In vitro, the killing effect on human transitional bladder carcinoma cell of iNOS will be enhanced by recombinant adenovirus rAd-iNOS combined with low dosage of chemotherapy like HCPT or Pirarubicin. This study demonstrated experimental evidence for iNOS gene therapy for human transitional bladder cell carcinoma combined with chemotherapy...