Study On RAAV-HSV-TK Gene Therapy Of Bladder Cancer With GCV

Bladder carcinoma is the most common urological malignant diseases in China.According to statistics, about 75% cases was superfic bladder cancer including 95%high risk. If without any drug treatment after Operation on high risk superfic bladdercancer, recurrence rate would be 60%-90% in the near future. The treatment for turnoris constitute of operation, radiotherapy and chemotherapy, which essentially viewfrom the angle of killing and wounding tumor cell first-hand. However, the tumorcells can not be drastically eliminated but normal tissue injuried easily. Immunesystem which occupies important position in anti-minor defence mechanism isdamaged, and especially cell immunity is impacted. With the development ofmolecular biology and gene engineering, biological treatment for tumor is beingthought highly of increasingly with favourable application prospect and becomes thefourth tumor therapy mode. Gene therapy is one of biological treatment. Approaching20 years phylogeny, it is the most potential. By the end of 1996, 2103 patientsaccepted gene therapy in the whole world, including 68.8% malignant tumor patients.In suicide gene treatment payed close attention to by numerous scholars, geneengineering is used to transduce suicide gene into tumor cells. Suicide gene includedin virus or bacterial genome, encodes special enzyme. Nontoxic prodrug can bemetabolized by the latter, become venenous production and make cell died.Therefore, the treatment is called "virus oriented enzymolysis prodrug medication" or"molecular chemotherapy". Thymidine kinase gene treatment is one of suicide genetreatments. The enzyme metabolizes NA and NA becomes diphosphorylation. Thelatter becomes triphosphorylation against rumor and selectively metabolizes GCV.GCV becomes triphosphorylation and then interferes and intercepts DNA normalsynthesis and cellular proliferation. In the treatment for tumor, herpes simplex virus-thymidine kinase gene is considered to be the most effective and also licensedinto clinical trial only. It can play a part in preventing DNA chain elongating, so thatselectively kill and wound suicide gene cells in multiplicative division period.Eukaryotic expression vectors mediating HSV-TK contain Retrovirus, Adenovirus,Adeno-Associated Virus and so on. However, we can also directly transduceplasmid nakedDNA without vector. In various kinds of studies about HSV-TK gene invivo and vitro, HSV-TK can play different roles because of different transgenosismethods.The topic utilizes rAAV-HSV-TK and technology about cell culture and animalbreed to kill and wound bladder cancer cells and to inhibit bladder cancer in nudemice. The objective of the study is that it can establish rationale for serial studies onsuccedent orthotopic animal model in vivo and tumor gene therapy including two ormore purpose genes.Aother objective is that it can open up a new study platform fortumor treatment, including the treatment for bladder cancer.Part 1Study on rAAV-HSV-TK gene therapy ofbladder cancer in vitro with GCVObjective: 1. Observe the effect of gene transfection T24 bladder cancer cells toprove its feasibility. 2. Utilize rAAV-HSV-TK to affect T24 bladder cancer cells, andthen observe the effect on apoptosis, growth of cell and time-dose dependent.Methods: 1. T24 bladder cancer cells were transfected with EGFP mediated by rAAVin vitro to obtain MOI of rAAV. 2. T24 bladder cancer cells were transfected withHSV-TK mediated by rAAV in vitro to prove its feasibility. 3. T24 cells were seededin 96-well tissue culture plates. Respectively add rAAV-HSV-TK and rAAV-MCSwith GCV of different concentration. The control group is without GCV. Carry out MTT experiment. Observe the dose dependent of rAAV-HSV-TK/GCV effecting onT24 cells. 4. T24 cells were seeded in 24-well tissue culture plates: Respectively addrAAV-HSV-TK and then add GCV of different concentration every 2 days accordingto the result of dose dependent. Carry out acridine orange/EB experiment. Accountthe number of them respectively every day, The control group is without GCV.Observe the time dependent of rAAV-HSV-TK/GCV effecting on T24 cells. 5. Decidethe apoptosis of T24 cells by flow cytometry. 6. In the group of rAAV-HSV-TK,10μg/mL GCV is added into T24 cells for 24, 48, 72hours and then use flowcytometry to detect cell cycle and DNA content respectively. Observe the effect ofcell cycle about rAAV-HSV-TK/GCV system.Results: 1.Optimization MOI is 1×106 v.p./cell. 2.Genome was successfullyextracted from T24 cells infected by virus. The product of PCR proves that rAAV hasbioactivity and can transfect T24 cells. 3. Genome was successfully extracted fromT24 cells transfected by HSV-TK. The product of PCR proves that T24 cellstransfected by HSV-TK can carry TK gene stably. 4.With concentration or action timeof GCV increasing, T24 cells survival descends drastively. Visible time and dosedependent was proved. 5. rAAV-HSV-TK/GCV system can make T24 cell apoptosis.6. T24 cells stop in G1 period from 24to 48 hours, but stop in S period from 48 to 72hours and obviously show deuto-G1 period, which implies about 10% tumor cellsappear apoptosis.Part 2Study on rAAV-HSV-TK gene therapy of bladdercancer of nude mice in vivo with GCVObjective: To construct bladder cancer animal model using nude mice, and thenstudy the therapeutical effect of rAAV-HSV-TK/GCV on the model in vivo to observe the effect of anti-tumor.Methods: 1. Cell suspension of T24 cells were subcutaneously injected into thearmpit of nude mice. The number of injected cells were 4×10~6 and then the nude micewere raised under SPF condition. To observe the xenograft tumor growth. 2. Bearingtumor nude mice were divided into 3 groups randomly: control group, rAAV-MCS group and rAAV-HSV-TK group. 4 weeks later of treatment, the nude mice werekilled by dislocating vertebrae cervicales. The volume of xenograft tumor wasmeasured. Fix the xenograft tumor for HE stain. The tissues of heart, liver, spleen,lung and kidney were also fixed for HE stain.Results: 1. After about 3 weeks, Nude mice show visible tumor on the injectedlocation. The rate of tumor formation is 90%. 2. We measured the tumor of nude miceevery 3 days. About 9 days later, the volume of rAAV-HSV-TK group was 0.71±0.11cm~3, 1.27±0.13 cm~3 for rAAV-MCS group and 1.24±0.17cm~3 for control group.Statistical analysis: Compare with rAAV-MCS group or control group, therAAV-HSV-TK group is notablely different (p＜0.05). 3. HE stain confirmed the tumor.There are no obviously cell degeneration and necrosis in the tissues of heart, liver,spleen, lung and kidney of each group.Conclusions: Experiment in vitro and vivo indicated that interested gene couldexpress in host cell mediated by rAAV and that the rAAV-HSV-TK with GCV couldinhibit the growth of bladder cancer effectively.