The Construction Of MGM-CSF Gene Modified Lung Cancer Cell Vaccine And Research Of Its Effects In Animal Model

PartⅠ: Construction and identification of mGM-CSF gene modified lung cancer cell vaccine@@@@Purpose: To construct the lung cancer cell vaccine modified by mGM-CSF gene and identify the expression of mGM-CSF in tumor cells. Methods: A pIRES2-EGFP-mGM plasmid, harboring a mGM-CSF gene and a EGFP gene, was designed and constructed by routine molecular cloning techniques. DNA sequence analysis and restrict digest were performed to confirm that EGFP tagged mGM-CSF gene was correctly inserted into pIRES2 vector, pIRES2-EGFP-mGM plasmid was further transferred into LA795 cells, and the expression of EGFP and mGM-CSF genes was examined by fluorescent microscopy and ELISA methods respectively. Lung cancer cell vaccine was prepared by irradiation with 100 Gy 137Cs. Results: Restriction analysis showed that the structure of pIRES2-EGFP-mGM plasmid was exactly the same as anticipated. Further result indicated that both EGFP and mGM-CSF genes were simultaneously expressed in LA795 cells. The expression level of mGM-CSF was suitable for preparation of the lung cancer cell vaccine.@@@@Conclusion: A plasmid has been established as the vector for construction of the mGM-CSF-secreting tumor vaccines, in which EGFP gene can serve as a reporter gene reflecting the expression of mGM-CSF gene. Murine lung cancer cells transfected with mGM-CSF gene could secret high level recombinant protein and can be used as tumor vaccine.@@@@PartⅡ: Research of the effects of mGM-CSF gene modified lung cancer cell vaccine in animal model@@@@Purpose: To investigate the non-specific anti-tumor immunity and mechanisms of immune responses induced by mGM-CSF gene modified LA795 lung cancer cell vaccine in animal models. Methods: T739 mice were immunized with LA795 lung cancer cell vaccine secreting mGM-CSF, meanwhile, the experiment animals injected with non-mGM-CSF secreting cancer cell vaccine and PBS were used as control groups. Changes of serum level of IFN-γwere tested by ELISA method. Flow Cytometry was employed to detect the phenotype of peripheral blood lymphocytes. The cytotoxicity of splenocytes against specific tumor cells was tested through LDH method. Results: The results showed that the serum IFN-γlevel and the percentage of CD8~+ T cells in mGM-CSF secreting LA795 lung cancer cell vaccine group are significantly increased comparing with the other two control groups(P<0.01), and the cytotoxicity of splenocytes against specific tumor cells was also significantly increased, which was much higher than the two control groups(P<0.01). Conclusion: mGM-CSF secreting LA795 lung cancer cell vaccine could induce T cell to differentiate and proliferate into CTL. The proliferation of CTL is the basis of specific anti-tumor immunity reaction induced by this vaccine, while the secretion of mGM-CSF significantly enhanced the immunologic effect of this vaccine, and the mechanism involved is mainly that CD8~+ CTL cells were directly activated.