Construction Of Bladder Tumor Vaccine Based On Dendritic Cell And Detection Of Its Biological Effect On Bladder Cancer

Bladder tumor, the most frequent urinary malignancy in Chinese people, of which most is non-muscle invasive tumor is characterized of high recurrence, owing probably to the local immunodeficiency in mucous membrane of urinary bladder, which makes residual tumor cells be able to escape from immunological surveillance, surviving and forming recurrence. Therefore, intravesical chemotherapy or immunotherapy is used postoperatively for the prevention of tumor recurrence and progress usually. Bacillus Calmette-Guerin (BCG) is currently the most effective intravesical immunotherapeutic agent, which takes the anti-tumor effect by the mechanism of effective activation of local immune response in mucous membrane of urinary bladder. However, treatments like intravesical BCG therapy are still not able to completely control recurrence and progress of bladder cancer. Prophylaxis of recurrence and progress of bladder cancer is a tough problem unsettled.Dendritic cells(DC) are believed to have the most powerful antigen presenting capacity(APC) and the native T cells can be only primed by DCs. But in tumor tissue and peripheral blood of tumor patients, amount of DCs decreases, of which maturity and function is inhibited. With the development of technique of DCs amplification vitro and DC vaccine constructed, Dendritic cell-based vaccine manifest a good clinical applications as a representative of new vaccines.Cyclooxygenase 2 (COX-2) is a kind of enzyme which is almost not expressed in normal human urothelial cells, but it is significantly over expressed in human multiple cancer cells. COX-2 affects cell differentiation, apoptosis, vascularization of tumors, and is related with tumor grading and staging, tumor progress and prognosis. Therefore COX-2 has been supposed to be a new therapeutic target for the treatment of bladder cancer. Many studies,with which it was found that COX-2 inhibitor could restrain progress of bladder cancer and had powerful entrainment on DCs, were carried out by our group. CpG, a DNA frag abstracted from Bacillus Calmette-Guerin Recent which unmethylated CpG motifs in bacterial DNA, was believed that had powerful immunostimulatory activity. Synthetic oligodeoxynucleotides(ODNs) containing CpG motifs have the same activity as bacterial DNA. CpG-ODNs are recognized by specific Toll-like receptor 9 on the surface of DCs and induce an activataion phenotype of DCs characterized by the expression of costimulatory molecules, enhanced antigen presentation to T cells.In recent years, studies about the relation of COX-2 , DC, CpG-ODN and bladder cancer were progressed by our group, constantly. The purpose of this study is to construct bladder tumor vaccine based on DC which is inducted by COX-2 inhibitor and CpG-ODN and detect its biological effect on bladder cancer. It should be a experiment foundation of looking for the new method of bladder cancer therapy and prophylaxis.Part I. Construction of bladder tumor vaccine based on DC and detection initially of its biological effect.Purpose: To construc bladder tumor vaccine based on DC and detect its biological effect, initially.Methods:1. Mononuclear cells were isolated from human cord blood and induced to differentiate to dendrtic cells (DCs) in vitro. Then DCs were stimulated mature using COX-2 inhibitor and CpG-ODN. Then the morphous and surface markers were detected.2. DCs were co-cultured with reagent concerted. Th1-type cytokine IL-12 and Th2-type cytokine IL-10 were measured in culture supernatant of each group by enzyme-linked immunosorbent assay (ELISA).3. Allogenic mixed lymphocyte reactivity (MLR) was determined by MTT assay of DCs in each group.Results:1. Results of identification showed that the cultured cells were morphology- and phenotype-typical DCs.2. Results of cytokine detection demonstrated that after BCG infection, both IL-12 and IL-10 production of DCs increased significantly compared with blank group, and no dominant immunological drift of the Th response occured. Celecoxib stimulated increased IL-12 and decreased IL-10 production. The results indicated that Celecoxib induced Th response drift to Th1, meanwhile CpG-ODN increased IL-12 and unchanged IL-10 production.3. Results of MLR detection showed that experimental group had higher stimulating index than control group.The effect of antigen presentation and inducing immune response of DC vaccine has been enhanced by combining celecoxib and CpG-ODN.Part II: Immunotherapy Effects of dendritic cell vaccine on bladder tumor cell T24 in vitro.Purpose: To investigate immunotherapy effects of dendritic cells vaccine on bladder tumor line T24 in vitro.Methods:1. The cytotoxic T lymphocyte activited by DC vaccine was prepared. Meanwhile as the target cell, bladder cancer cells T24 were resuscitated and cultivited.2. Adherent T24 cells were respectively co-cultured with the DCs. Cytotoxicity of CTLs activted by DCs of each group was measured by LDH release assay.Results:1. As the target cell, bladder cancer cells T24 were resuscitated and cultivited, successfully. And The cytotoxic T lymphocyte activited by DC vaccine were gained.2. Results of LDH release assay suggested that DC vaccine activated CTLs enhanced cytotoxicity on human bladder cancer cell line T24.Partâ…¢Immunotherapy Effects of dendritic cell vaccine plused with tumor antigen on bladder tumor in vivo.Purpose: To investigate immunotherapy effects of dendritic cells vaccine plused with tumor antigen on bladder tumor in T739 mice.Methods:1. After the BTT739 bladder tumor model in T739 mice were successfully established, DC vaccine were injected respectively into the margins of the tumor(peritumoral) on day 7 and day 14 after tumor cells inoculation. Tumor weight, volume and the living time of mice were recorded.2. After two mice cured with DC vaccine were rechallenged with BTT739 cells, the development of tumor was monitored.Results:1. Tumor weight and volume inhibition effects of DC vaccine treatment experiment group were significantly higher than that of control group (P<0.01). The living time of experiment group mice was significantly longer than that of control group. DC vaccine plused with tumor antigen can inhibit tumor growth and prolong the living time of T739 mice with BTT739 bladder tumor.2. Two mice cured with DC vaccine were tumor-free 30 days after being rechallenged with BTT739 cells. DC vaccine plused with tumor antigen can lead to complete rejection of tumor rechallenge and provide long-term maintenance of tumor immunity, It should be an appealing immunotherapeutic approach to preventing the recurrence of bladder tumor in clinic.Summary and Conclusions:1. Mononuclear cells were isolated from human cord blood and induced to differentiate to dendrtic cells (DCs) in vitro. Then DCs were stimulated mature using COX-2 inhibitor and CpG-ODN and were confirmed that the cultured cells were morphology- and phenotype-typical DCs. Meanwhile Th response drifting to Th1 was induced.2. DC vaccine can induce effector cells to appear strong cytotoxicity and killing activity on bladder cell T24.3. DC vaccine plused with tumor antigen can inhibit tumor growth and prolong the living time of T739 mice with BTT739 bladder tumor.4. DC vaccine plused with tumor antigen can lead to complete rejection of tumor rechallenge, which shows that this method should be an appealing immunotherapeutic approach to preventing the recurrence of bladder tumor in clinic.