Study On The Effect Of Anti-bladder Cancer Induced By DC Vaccine Loaded With Tumor Antigen In Vitro And In Vivo

1. To investigate the expression of tumor associated antigen(TAA) in tissues of bladder cancer, adjacent tissues and normal tissues of multiple organ.2. To investigate a method of establishing human bladder cancer-bearing composite animal model with reconstituted human immune system, and to evaluate the model.3. To investigate the way to prepare DC vaccine loaded with bladder cancer antigen and the activation of homologous T lymphocytes in vitro, further to observe the specific killing effect of bladder cancer cells induced by T lymphocytes.4. To investigate the specific antitumor effects against orthotopic transplantation tumor of mice model with human bladder cancer in vivo, induced by DC vaccine loaded with bladder cancer antigen. Methods1. Expression detection of TAA in human bladder transitional cell carcinoma (BTCC) tissues, adjacent tissues and normal tissues of multiple organs: Expression of TAA combined with antihuman bladder carcinoma monoclonal antibody BDI-1 in BTCC, adjacent tissues and normal tissues of multiple organs were identified by tissue microarray technology using immunohistochemical Elivision method. The positive expression rate and positive intensity of TAA were analyzed and compared among three different pathological grading of BTCC, between the bladder cancer tissue and adjacent tissues.2. Establishment and identification of human bladder cancer-bearing composite animal model with reconstituted human immune system:Human peripheral blood mononuclear cells(hu-PBMCs) were isolated by density gradient centrifugation. Balb/c-nu mice model with reconstituted human immune system was established by intraperitoneal injection of hu-PBMC. Phenotype of human CD3+T lymphocytes and CD19+B lymphocytes in murine peripheral blood were detected by Flow Cytometry. The level of human IgG in Balb/c-nu mice blood-serum was measured by Enzyme-linked Immunosorbent Assay(ELISA). Immunohistochemistry was used to detect infiltration of human CD3+T lymphocytes and CD19+B lymphocytes in the liver and spleen of mice. The experimental results of immune reconstitution group were compared with non-humanized group. Balb/c-nu mice composit model with reconstituted human immune system and bearing subcutaneous transplantation tumor of human bladder cancer was established. Tumor growth curve was drawn and survival rates of mice were calculated. The experimental group was compared with tumor group without reconstituted human immune system.3. Construction of DC vaccine loaded with EJ cell lysate antigen and the effect of anti-bladder cancer induced by DC vaccine in vitro:EJ cell lysate antigen components were obtained by freeze-thaw method. The cell lysate protein content was determined by BCA protein quantitative method. Hu-PBMCs were isolated by density gradient centrifugation and were cultured in vitro. The dendritic cells(DCs) were induced by rhGM-CSF, rhIL-4, TNF-αfrom hu-PBMCs cultured in vitro. The phenotypes of the cultured DCs were identified by FCM. EJ cell lysate antigen pulsed DC. DC vaccine loaded with EJ cell lysate antigen was generated. CD3÷T lymphocytes in spleen tissue in Balb/c-nu mouse with reconstituted human immune system were isolated by T-immunomegnetic beads. The proliferation ability of autologous T cells and CTL's cytotoxicity towards EJ cells induced by DC vaccine was detected.4. The specific effects of anti-bladder cancer induced by DC vaccine loaded with tumor antigen in vivo and monitored by fluorescence imaging system:EJ cells were infected by lentivirus carrying green fluorescent protein(GFP) gene. GFP gene was transfected into EJ cells. The stable highly expressing GFP cell lines were established. EJ cells expressing GFP were perfused into bladder of Balb/c-nu mice with reconstituted human immune system by puncturing method. Human bladder cancer-bearing composite and visual animal model with reconstituted human immune system was established. After DC vaccines were injected intraperitoneally in mice, the growth of tumor was dynamically observed by fluorescence imaging system in vivo. The survival rates of mice were calculated in the experimental group and control group. The level of IFN-γin peripheral blood of Balb/c-nu mice was measured by ELISA. The infiltration of human T lymphocytes and mature DC in the spleen and tumor tissues of mice was detected by FCM.Results1. Expression of TAA in human BTCC tissues, adjacent tissues and normal tissues of multiple organs:The positive staining was in the membrane of cells. In the G1,G2,G3 pathological grades of BTCC, the positive expression intensity of TAA combined with BDI-1 had no statistical difference(P>0.05). Compared with adjacent tissues, the positive expression intensity of TAA in BTCC was significant higher. The difference was statistically significant(P<0.05). In tissues of normal organs, the positive expression of TAA was observed in esophagus, cervix, gastric, colon, small intestine, prostate, pancreas, lung, thymus and breast tissues.2. Establishment and identification of human bladder cancer-bearing composite animal model with reconstituted human immune system:Human CD3+T lymphocytes and CD19+B lymphocytes cells were detected in peripheral blood and spleen of Balb/c-nu mice with reconstituted human immune system. The level of human IgG in mice with reconstituted human immune system was significant higher comparing with control group(P<0.05). The level of human IgG was also higher at the twelveth week after human immune system was reconstituted in Balb/c-nu mice. EJ cells were implanted subcutaneously in the mice with reconstituted human immune system and tumor formation rate was 100%. Tumor growth curve and survival rates in the experimental group was the same as control group(P>0.05).3. Construction of DC vaccine loaded with EJ cell lysate antigen and the effect of anti-bladder cancer induced by DC vaccine in vitro:The EJ cell lysate protein content was about lng/ml(103 EJ cells). The dendritic cells(DCs) were induced by rhGM-CSF, rhIL-4 from hu-PBMC. Tumor necrosis factor-a(TNF-α) promoted immature DCs transformed into mature DCs. The expressions of CD83 and CD80 on mature dendritic cells were statistically significant(P<0.05). T lymphocytes were activated by DC vaccine loaded with EJ cell lysate antigen. The stimulate index was higher, and the killing rate of CTL against EJ cells was 62.58±6.13%. Compared with control group, these difference were statistically significant(P<0.05).4. The specific effects of anti-bladder cancer induced by DC vaccine loaded with tumor antigen in vivo and monitored by fluorescence imaging system:The stable highly expressing GFP cell lines were established using lentivirus vectors. Compared with EJ cells, no obvious difference was found in cell growth curve, FCM detection, growth curve of subcutaneous transplantation tumor and survival time of mice(P>0.05). Fluorescence signals were detected in EJ-GFP cell subcutaneous transplantation or orthotopic transplantation tumor by fluorescence imaging system in vivo. Orthotopic transplantation tumor grew slowly in mice after DC vaccine intraperitoneal injection. Survival time of mice in experimental group was prolong. Compared with control group, the difference was statistically significant(P<0.05). CD3+,CD4+,CD8+T lymphocytes and mature DC were detected in mice spleen and tumor tissues. The level of IFN-y was higher in mice peripheral blood. Compared with control group, the difference was statistically significant(P<0.05).Conclusion1. Expression of TAA in human BTCC tissues, adjacent tissues and normal tissues of multiple organs:1) The positive staining of TAA combined with BDI-1 is mainly in the membrane of cells. The positive expression intensity of TAA combined with BDI-1 in BTCC tissues is higher than that in adjacent tissue.2) In the three different pathological grades of BTCC, the positive expression intensity of TAA combined with BDI-1 has no statistical difference. In normal tissues of multiple organs, the positive expression of TAA is observed.3) It is an efficient and rapid method to detect TAA by tissue microarray technology. Tissue microarray technology is an useful tool for the detection of TAA.2. Establishment and identification of human bladder cancer-bearing composite animal model with reconstituted human immune system:1) Balb/c-nu mice model with reconstituted human immune system can be established by intraperitoneal injection of hu-PBMC. The human T and B lymphocytes can migrate normally and live for a long time in mice body, and can maintain certain biological activities (such as secreting human IgG, etc), which provides guarantee for human lymphocytes to play an effective immune response in mice body. 2) Balb/c-nu mice composite model with human bladder cancer-bearing and reconstituted human immune system is useful for the immunotherapy research of human bladder urothelial carcinoma.3) In this research, human CD3+T lymphocytes and CD19+B lymphocytes are rare in mice liver. Distribution and migration of human lymphocytes is not random. Human lymphocytes settle in subprime lymphopid organs of mice selectively.3. Construction of DC vaccine loaded with EJ cell lysate antigen and the effect of anti-bladder cancer induced by DC vaccine in vitro:1) The mature DCs can be induced by rhGM-CSF, rhIL-4 and TNF-αfrom hu-PBMCs.2) EJ cell lysate contains human bladder cancer antigen composition. DC vaccine can be constructed by loading EJ cell lysate antigen composition.3) The proliferation ability of autologous T cells and CTL's cytotoxicity towards EJ cells can be induced by DC loading with EJ cell lysate.4. The specific effects of anti-bladder cancer induced by DC vaccine loaded with tumor antigen in vivo and monitored by fluorescence imaging system:1) The EJ cell lines expressing GFP(EJ-GFP) can be established in vitro by lentivirus vectors carrying GFP gene. The expression of GFP is stable, highly and long time. The Balb/c-nu mice with reconstituted human immune system are inoculated with the cell lines. The visual model with hunan bladder urothelial cancer subcutaneous transplantation or orthotopic transplantation tumor and reconstituted human immune system can be established successfully.2) DC vaccine loaded EJ cells lysate antigen composition can induce the specific effects of anti-bladder cancer by intraperitoneal injection in vivo. DC vaccine can inhibit tumor growth and obviously prolonged the exist time of animals bearing tumor. The immune response may be related with the infiltration of human CD4+, CD8+T lymphocytes and mature DC in the tumor tissues, or IFN-γcontent increasing in peripheral blood of Balb/c-nu mice.3) The orthotopic transplantation tumor growth of mice can be monitored by fluorescence imaging system noninvasively and real time dynamically. The specific effects of anti-bladder cancer induced by DC vaccine can be evaluated objectively.