Study On The Anti-Cancer Effect Of EJ Sensitized DC Vaccine On Human Immune Reconstruction NOD/SCID Mice Model Bearing Human Bladder Carcinoma

Objective:To establish human immunological characteristics model with NOD/SCID mice. Preparation of EJ cell lysate-pulsed DC vaccines will be applied to treat the mice model bearing human bladder carcinoma for bladder cancer, and then analysing and evaluating its biological effects, all of which would provide a new idea and theoretical basis to bladder cancer.Methods:First:To establish and identify human immune reconstruction of NOD/SCID mouse model1 Sixteen normal mice after screened were randomly divided into 2 groups:in the experimental group, each was given intraperitoneal injection of 0.5 ml human PBMC 4×107, while in the contral group 0.5 ml sterile PBS.2 The model were identified in biological and immunological characteristics: Biological characteristics; Peripheral blood, spleen and liver were colected in the first 4,8 and 12 weeks of treatment in 2 groups; Detecting human CD3+T and CD19+B lymphocytes in the mice blood by flow cytometry and detecting the infiltration of human CD3+T and CD19+B lymphocytes in mice spleen and liver by immunohistochemical staining; Measuring human IgG protein content in the mice blood.Second:Study the anti-tumor effect of EJ pulsed DC on the human immune reconstruction NOD/SCID mice model bearing human bladder carcinoma1 In vitro, combining with cytokine rhGM-CSF, rhIL-4 and TNF-a to have an effect upon mononuclear cells that came from human peripheral blood and to induse differentiation of DC cells, then we identified the DC cells by its morphology and surface specific markers; After 4 weeks of Intraperitoneal injection of DC vaccine to the NOD/SCID mice model, spleen was colected to selecte human T lymphocytes through magnetic cell sorter; Mesuring the proliferation of T cells activited by EJ pulsed DC vaccine with [3H]-TdR incorporation test and evaluating T-cell cytotoxicity activited by DC in two groups(no load and load EJ lysate-pulsed DC) through 51Cr release test.2 After 24 hours of reconstructing human immune, NOD/SCID mice was seeded EJ cells and devided radomly into two groups in 10 days:EJ-DC group,10, each was intraperitoneal injected the EJ-pulsed DC vaccine 0.2 ml(including 2×105 EJ lysates DC cells); DC group,10, non EJ-pulsed DC were given in the same way and with the same amount. We observed and recorded biological characteristics of the mice including vital signs and growth of the tumor; Using ELIS A to test the serum concentrations of IFN-y after treatment; Detecting the infiltration of T and DC cells in the mice liver through Immunohistochemistry and in the mice tumor and spleen through flow cytometry, and observing the pathaology of tumor with Histopathology.Results:First:1 CD3+T and CD19+B lymphocytes in peripheral blood of the mice who have been seeded human monocytes for 4 weeks were respectively 85.6% and 76.7% of all monocytes.2 Immunohistochemistry results showed that there were CD3+T and CD19+B lymphocytes in spleen of the mice.3 The amount of IgG in serum of the mice after 4,8 and 12 weeks of transplantation was respectively 863μg/ml,1217μg/ml and 958μg/ml.Second:1 The proliferation index of autologous T-lymphocyte after activated by EJ-pulsed DC and non EJ-DC were respectively 7.2,1.0, and the killing rate of CTL activited by EJ-plused DC and non EJ-DC were respectively 63.1±5.7%,21.5±4.8%, The difference was statistically significant(p<0.05).2 All mice were got tumors.Compared with DC group, the EJ-DC group survival a longer time and have a lower increasing in tumor volume(p< 0.01).3 1,2 and 3 weeks after treatment, serum IFN-y levels in EJ-DC group were respectively 19.6,26.8,23.7 IU/ml while in DC group 7.2,13.5,15.6 IU/ml, The difference was statistically significant(p<0.01).Compare with DC group, there were more CD4+, CD8+ T lymphocytes as well as DC-cell infiltration in spleen and tumor tissue in the EJ-DC group, but no different in the mice liver. Pathology confirmed for bladder cancer tumors in mice.Conclusion:1 Human immune reconstraction NOD/SCID mice model can be successfully made by intraperitoneal injection of human peripheral blood lymphocytes, which would lay a good foundation for further study.2 DC precursors could be obtained from PBMC in human peripheral blood and the combination with rhGM-CSF, rhIL-4 and TNF-a could induce a large number of typical DC cells.3 In vitro, EJ pulsed DC vaccines could activate stronger anti-tumor effect; and could slow down the rate of tumor growth and prolong survival in NOD/SCID mice model.