Experimental Study On Adenovirus-mediated TK/GCV Gene Therapy In Combination With Tumor Necrosis Factor-α (TNF-α) For Murine Bladder Cancer

Background Bladder carcinoma is not only one of the commonest cancers of urothelial system,but also an important problem of public health Bladder carcinoma is the fourth common malignant tumors in America,which is first morbidity of genitourinary system in our country.There are more than 200,000 patients diagnosed with bladder carcinoma and about 12,000 patients died of bladder carcinoma.Transitional epithelial cell cancer accounts for 90%of bladder carcinoma. the prostecdtive efficacy of therapy for bladder carcinoma is not satisfactory. Because of the unique bionomics of bladder carcinoma,the effect of surgical therapy, radiotherapy and chemotherapy for bladder carcinoma is quite limited.However there is high recurrence rate after the surgery,espcially advanced stage tumor and carcinoma of bladder in situ as tolerated by the patient.The recurrence rate is 60%in five years and 88%in fifty years,and 10%to 30%of palindromia tumor progressed infiltrating type,the prostecdtive efficacy isn't very good.The high recurrence rate of superficial bladder carcinoma after transurethral resection is one of therapeutic problems urgent to be solved.With the development of studies on the suicide gene therapy for bladder carcinoma,we realized that suicide gene therapy is a promising management for bladder carcinoma.TK/GCV system is optima extensive suicide genic system at present.It can express thymidine kinase that make asepsis prodrug such as ganciclovir phosphorylation.The phosphorylated ganciclovir effect dividing phase cells by incorporate cell's deoxyribonucleic acid catenation as substrate and it can inhibit cell deoxyribonucleic acid pclymerase so that the cell deoxyribonucleic acid synthesize is terminated and cell is dead.There is considerable scholar utilize retrovirus or adenovir to mediate TK/GCV suicide genic system to take various kinds of tumorous therapy investigation,the role effect is manifest.However,there are also shortcomings in gene therapy such as low gene transfection efficiency,poor targeting of gene transduction,toxicity and harm of genes and genetic carriers on human body,the autoimmune reponse resulted from the carrier and the killing of tumor cells,additionally,the metabolic product of prodrug only has lethal effect on the cells in dividing phase.In order to make up the disadvantages of suicide gene therapy,more and more scholars dedicate themselves to the study on the combination of present therapy and suicide gene therapy to search for a new treatment for the recurrence of bladder carcinoma.Tumor necrosis factor-αis a kind of cell factor with many bioactivity,especially to many species of tumour cells,it can induce straight lethal effect and mediate containment.In vitro,it can induce direct lethal effect to tumor cells with cell toxic action in hypsi-concentration,with low density it can reach the lethal effect through which induced apoptosis of non-multiplication period cells.In vivo,it can enhance immunological effect by immunological regulation effect which derivn the production of other cytokine such as interleukin-1,interleukin-2 and interferon-γetc.it educe anti-tumor effect by enhanced cytotoxicity of cell natural killer cell and macrophage;it evoke Tumor necrosis by induce region Inflammatory reaction of tumor organism,which result in blood coagulation,ischemia and cellular necrosis.There isn't correlated report of the way,which thymidine kinase gene in combination with tumor necrosis factor-αtherapy to carcinoma of bladder at present. In this research,we study the lethal effects of tk gene in combination with low doses of tumor necrosis factor-αon murine bladder carcinoma cell line MB49 cells and compare the differences in effects between each therapy in order to provide certain experimental bases for the suicide gene therapy in combination with cell factors for bladder carcinoma.Methods1,The MB49 cells and HEK293 cells were cultured in vitro.2,The 293 cells were transfected with replication defective adenovirus containing TK gene and green fluorescent protein(GFP) gene,then the adenovirus was amplificated and adenovirus titer was determined by quantifying the GFP positive cells.3,The MB49 cells of murine bladder carcinoma were trasfected with TK gene to obtain the MB49 cells transfected with TK gene.After the transfection of murine bladder carcinoma cell line MB49 cells with the TK adenovirus,extract total Rna of MB49 cells and the infected MB49 cells to carry out RT-PCR and was indentified by electrophoresis.4,The MB49 cells infected with TK gene were cultured in vitro with different concentrations of GCV.The concentration of GCV was 0,5,10,12.5,25,50, 75,100,125μg/ml respectively.The MB49 cells were cultured in vitro with different concentrations of TNF-α.The concentration of TNF-αwas 0,2.5,5,10, 20,25,50,80,100μg/ml respectively.The cell survival rates when murine bladder carcinoma cells were affected by GCV and TNF-αalone for 72h were observed by MTT assay.5,According to previous experiment result and effective concentration of GCV and TNF-αfrom related experiment ahead,we chose the medicine concentration that make minor lethal effect to MB49 cells(GCV use 0,5,10,25,50μg/ml,TNF-αuse 0,5,20μg/ml ) fractionate GCV group and GCV in combination with TNF-αgroup,which add to MB49 cells infected with TK gene,and MB49 cells as blank group.The cell killing rates of trasfected carcinoma of bladder treated by different group were observed by MTT assay.The apoptosis of different group were detected by flow cytometry(FCM).6,The animal models were established by subcutaneously injecting about 1×10~6 MB49 cells on the 30 C57BL/6 female mice.Two weeks ago 24 mice with about 100 mm~3 of the tumor volume were selected,and divided into the 4 groups randomly:①The control;②TNF-αinjected subcutaneously around the tumor;③GCV injected intraperitioneally;④TNF-αinjected subcutaneously around the tumor +GCV injected intraperitioneally.The adenovirus was injected intratumorally with the dose of 100μl/d/mouse/time×3 days.During the course of the treatment,the volumes of the tumors were measured(volume=width~2×length×0.5236).The tumor tissue were analyzed by histopathology.7,The datas were dealed with SPSS13.0,analysed by One-Way ANOVA and Univaiate,P≤0.05 as statistical significance.Results1,The recombinant adenoviruses were successfully amplified in HEK 293 cells and its titer was about 2×10~9 PFU/ml after purified.2,The RT-PCR assay showed that the adenovirus-transfected MB49 cells expressed TK mRNA.4,The survival rate of GCV on adenovirus-transfected MB49 cells decreased gradually as the concentration increased.the rate was(96.97±1.67)%when the concentration was 5μg/ml,the rate decreased to(80.82±0.64)%when the concentration was 25μg/ml and the rate was(28.15±1.61)%as the concentration was 125μg/ml,The survival rate of TNF-αon MB49 cells decreased gradually as the concentration increased.It showed that can inhibit the growth of murine bladder carcinoma cells with concentration dependent.4,When GCV in combination with low concentration of TNF-αwere added, the killing rate of the infected murine bladder carcinoma cells in each group increased significantly(P=0.000),compared to the group added GCV alone.The killing efficiency in therapeutic alliance group increased obviously when the concentration of TNF-αwas higher.The killing rate of the gourp added 50μg/ml GCV alone was(24.39±1.10)%in 72h,The killing rate was(40.05±0.97)%of the group added GCV(50μg/ml) in combination with TNF-α(5μg/ml) and the rate was (65.47±0.67)%of the group added GCV(50μg/ml) in combination with TNF-α(20μg/ml).It indicated that the lethal effets of TK/GCV in combination with low doses of TNF-αon the murine bladder carcinoma cells were better than those of TK/GCV alone.We can detected the apoptotic peak of sub-G1 stage in group of TK/GCV alone,TNF-αalone and TK/GCV in combination with TNF-αby flow cytometry.The apoptotic rate of TK/GCV alone is(6.66±0.13)%and TNF-αalone is(7.76±0.12)%,while the apoptotic rate of the unification group reach (12.99±0.09)%.The poptotic rate increased significantly(P=0.000)。Both TK/GCV system and TNF-αcan induce apoptosis effectively,furthermore the two has joint action.5,When animal experiment in vivo completed,the average tumor volume of TK/GCV treated group was 93.43±2.10 mm~3,and TNF-αtreated group was 53.95±2.61 mm~3 smaller than that of the control proup significantly(171.52±4.33 mm~3)(P=0.000).The average tumour volume of TK/GCV+TNF-αtreated group was 18.23±1.11 mm~3,smaller than any others(P=0.000).6,through histopathology detection we can see all the therapy group emerged cell necrosis and apoptosis.The affiliation group had the better than utilize suicide gene and cytokine alone.There are few tumor cell survived in bouncary in pathological section of the affiliation groupConclusion1,TK gene can be inducted into the murine bladder carcinoma MB49 cells by adenovirus vector and express.2,GCV can inhibit and kill the MB49 cells infected with TK gene efficiently. It had dose-dependent relation.3,TNF-αcan inhibit and kill the MB49 cells.There is dose-dependent relation.4,TK/GCV suicide genie system in combination with low doses of TNF-αcan promote the apoptosis of the murine bladder carcinoma MB49 cells in vitro.5,TK/GCV suicide genie system in combination with low doses of TNF-αcan enhance the lethal effect of suicide gene on the murine bladder carcinoma MB49 cells in vitro.6,TK/GCV suicide genic system in combination with low doses of TNF-αcan enhance the lethal effect of suicide gene on the murine bladder carcinoma in vivo.