Study About Expression Of Smac To Control The Apoptosis Of Bladder Cancer Cell By Inhibiting IAPs

Part 1Cloning UP1b promoter gene from Chinese PeopleObjective : To clone Uroplakin 1b promoter gene from Chinese people.Methods : At first, we get human UP1b promoter gene sequence from literature and Genebank of NCBI. UP1b promoter gene primer was designed by primer premier 5.0 software. Then the molecular cloning method was used to extract total DNA from a blood sample of bladder TCC. Full length UP1b promoter gene was cloned from human genome DNA by polymerase chain reaction(PCR) and was purified. The UP1b promoter gene was identified by sequencing.Results: The nucleic acid sequencing results indicated that Chinese Uroplakin 1b promoter DNA (235 bp) was successfully cloned. The sequence analysis demostrated the cloned sequence is 99% homologous with overseas reported sequences by Genebank of NCBI(Accession number AF324865) and is 100% homologous with reported sequences by Olsburgh.Conclusion: Human Uroplakin 1b promoter gene was cloned from Chinese blood tissues. It established the basics for further exploring of gene treating bladder cancer. Part 2constructing pcDNA3.1 (-) UP1b promoter-Smac eukaryonexpression vectorObjective : To construct a new kind of recombinant plasmid containing human second mitochondria-derived activator of caspase (Smac) protein gene cNDA and Uroplakin 1b(UP1b) promoter gene.Methods : At first, we identified the pcDNA3.1(-)Smac plasmid by sequencing. Then the UP1b promoter gene was cloned from human genome DNA by polymerase chain reaction(PCR) and was purified and retrieved. To digest pcDNA3.1(-)Smac plasmid and UP1b promoter gene with Nhe1 and Xba1 endonuclease , we retrieved a big fragment from pcDNA3.1(-)-Smac and a 250bp fragment from UP1b promoter gene. Recombined the big fragment and 250bp fragment by T4DNA ligase. The recombination pcDNA3.1(-)UPlb promoter-Smac plasmid was identified by polymerase chain reaction(PCR) and sequencing.Results: We succeeded in constructed a sort of eukaryon expression plasmid vector With pcDNA3.1(-)-UPlb promoter-Smac. It was identified by sequencing that the length and sequence was same as that human being's.Conclusion: A new eukaryon expression plasmid with UP1b promoter gene and Smac gene has been constructed. It establishes the basics for gene treating bladder cancer. Part 3Construction of human UP1b promoter reporter gene plasmids and analysis of promoter activity of plasmidsObjective : To clone human Uroplakin 1b promoter gene and construct luciferase reporter gene plasmids pGL 3- Enhancer-UP1b promoter, and to analyze the promoter activity in human TCC cell line BIU-87 cells.Methods : At first,. The UP1b promoter gene was cloned from human genome DNA by polymerase chain reaction(PCR) and was purified and retrieved. To digest pGL 3-Enhancer plasmid and UP1b promoter gene with Nhe1 endonuclease , we retrieved a big fragment from pGL 3- Enhancer and a 250bp fragment from UP1b promoter gene. Recombined the big fragment and 250bp fragment by T4DNA ligase. The recombination pGL3-Enhancer-UPlb promoter plasmid was identified by polymerase chain reaction(PCR). Transient transfected pGL3-Enhancer-UP1b promoter plasmid into human TCC cell line BIU-87 cells by Lipofectamine 2000. Cells were be cultivated and luciferase assay were used.Results: We succeeded in constructed a luciferase reporter gene plasmids vector pGL 3- Enhancer-UP1b promoter. It was identified by PCR. Be observed by fluorescence microscope, The human TCC cell line BIU-87 cells which were transfected with pGL3-Enhancer-UPlb promoter plasmid expression luciferase activity.Conclusion: Human UP1b promoter luciferase reporter gene constructs were successfully constructed,. Human UP1b promoter has good promoter activity. Part 4The recurrence risk factors in patients with transitional call carcinoma of bladderObjective: To study recurrence factors and set up a model to evaluate the prognosis in patients with bladder cancer.Methods: A analysis on recurrence-related factors was made by Cox's proportional hazards model analysis and logistic multiple linear regression model analysis in 212 patients with transitional cell carcinoma that treated by operate from 1995-2001.These factors included clinical and pathologic figues.Results: The most impotant factor is metastasis to the regional lymph nodes,the Hazards ratio is 6.6(p=0.0004),follwed by multiple tumers(Hr=2.255,p<0.0001),tumer in trigone and bladder neck (Hr=2.053,p<0.0001), stage(Hr=2.057, p<0.0 001) ,grade(H r=1.569,p=0.0081), intravesical chemotherapeutic instillations (Hr=0.559, p=0.0011), hematuria(Hr=0.762,p=0.0076).A predicting equation was established ,and the predicting values were calculated according to the individual features of patients. The predicting and actual values were compared ,and the sensitivity, specificity and overall concordance were 83.5%,67.6%,80.1% respectively.Conclusions: The eveluation on prognosis could be made quite accurately based on these factors.