| Background： Renal cell carcinoma (RCC) constitutes90%of primary renalneoplasms, and has the highest lethality among urologic neoplasms. However,metastatic RCC is not sensitive to traditional treatments, such as chemotherapy,radiotherapy and cytokine therapy, and its prognosis is relatively poor. DNA vaccineis a new method of cancer immunotherapy which has been tested in multiplepreclinical experiments and clinical trials. We have developed a novel replicabledouble-plasmids DNA vaccine against renal cancer, which could kill renal carcinomacells as well as inhibit angiogenesis in tumor. We hope that our vaccine will provide anew method of immune therapy in renal cell carcinoma.Objective: To evaluate the pharmacodynamics of our novel replicabledouble-plasmids DNA vaccine against renal cancer, and investigate the possibleimmunologic mechanism. In order to investigate the antitumor efficacy, we establish amurine RCC cell line which stably expressing hG250antigen and luciferasesimultaneously.Method:1. The establishment of the Renca-hG250/Luc cell line: The plasmidpIRES-Luc-hyg was transfected into Renca-hG250cells. After screening culture byHygromycin B and limiting dilution approach, we established a murine RCC cell lineRenca-hG250/Luc. The expression of hG250antigen and luciferase gene wasidentified by Western blot, flow cytometry and bioluminescence imaging.2. Animal experiments for double plasmids vaccine: The Balb/c mice wereimmunized with double-plasmids vaccine and other control vaccines by intramuscularinjection and electroporation. Then2×105Renca-hG250/Luc cells were subcutaneously inoculated into Balb/c mice to establish tumor-bearing mice model.Then we observed the anti-tumor efficacy and study the possible immunogicmechanism of the double-plasmids vaccine. We detected the specific antibody inserum of immunized mice by ELISA assay, and detected the specific IFN-γ secretionof immunized mice’s spleen lymphocytes. Then the proliferation activity of spleenlymphocytes evaluated by CCK-8assay, and the secretion of Th1cytokins weredetected using ELISA assay. Finally, the specific CTL activity was also detedctedusing LDH assay.Results:1. A murine RCC cell line Renca-hG250/Luc stably expressing hG250and lucgene was established successfully, and the positive rates of hG250and Luc were81.87%and81.03%respectively.2. The immunological effect of the double-plasmids vaccine was investigated inBalb/c mice. The results showed that the double-plasmids vaccine significantlydelayed the growth of renal cell carcinoma in immunized Balb/c mice and increasedthe life span of tumor-bearing mice compared with the other control vaccine.Furthermore, the double-plasmids vaccine induced both humoral and cellular immuneresponses in Balb/c mice after immunization. The specific antibody targeting hG250and mVEGFR2could be detected in the serum of mice, and the ratio of IgG1/IgG2aof hG250antibody was0.75. Meanwhile in the double-plasmids vaccine immunizedmice, the spleen lymphocytes of presented a higher proliferation activity, and thenumber of specific IFN-γ secretion were405±52dots/5×105lymphocytes and453±61dots/5×105lymphocytes when stimulated with hG250or mVEGFR2antigenrespectively. And more Th1cytokines IL-2and IFN-γ were detected in the culturalsupernatant of lymphocytes. Finally, the cytotoxicity of spleen lymphocytes againstRenca-hG250/Luc target cells was51.12%,39.23%and25.88%at the effector-targetratio of40:1,20:1and10:1respectively.Conclusion:In summary, the novel replicable double-plasmids DNA vaccine against renalcell carcinoma induced higher humoral and cellular immune responses targeting hG250and mVEGFR2antigen. And it inhibited the growth of renal cell carcinomaeffectively through killing tumor cells and inhibiting angiogenesis. The researchprovided a new method of immune therapy in renal cell carcinoma. |
