Fare Lizozim ve İnterlökin-2 Genlerinin Klonlanması ve Malign Melanomada Anti-tümöral DNA Aşısı Olarak Kullanılması

ABSTRACTCloning of Mouse Lysozyme and Interleukin-2 Genes and Using asAnti-tumoral DNA Vaccine in Malign MelanomaAli İrfan GÜZELImmunocompetent hosts can potentially recognize malignancies andimpede their proliferation by killing them. However, any failure of the immunesystem due to the lack of tumor immunogenicity or a tumor inducedimmunosuppressive environment causes malignant cells to escape from theimmune detection. One goal of tumor immunotherapy is to manipulate the hostimmune response to the malignancies to allow for the recognition andelimination of them.The aim of this study is to clone mouse interleukin-2 (IL-2) and lysozymegenes to pHM6 mammalian expression vector and use them in B-16V melanomacells for the purpose of immunotherapy. In different studies made previously, IL-2 has been used both in the form of DNA vaccine and peptide, but lysozyme onlyin the peptide form. The lysozyme, known to have anti-tumoral andimmunomodulatory as well as antibacterial activities was investigated first timehere in the form of DNA vaccine.For this purpose, four experiments were made to evaluate the anti-tumorigenic effects of lysozyme and interleukin-2 genes when used in vaccineform. 1) Plasmids combined with mouse IL-2 and lysozyme genes (pHM6mIL-2and pHM6mLys) were injected in the form of DNA - liposome complex into thetumor mass formed by the injection of B-16V melanoma cells into mice (inbredC57BL/6) subcutaneously. 2) Recombinant plasmids were injected into micesubcutaneously two days before the injection of B-16V cells to the same site. 3)B-16V tumor cells transfected with recombinant plasmids (B-16VpHM6mIL-2 andB-16VpHM6mLys) were injected subcutaneously to the mice. 4) Tumor free miceof the assay 3 were challenged with B-16V melanoma cells.Overall 277 mice were used in 15 groups of four experiments. The resultsof experiments 1 and 2 showed that the injection of recombinant plasmidscarrying IL-2 and lysozyme genes either before or later the tumor developmentdid not significantly retard the progress. However, in experiment 3, the micewere remained tumor free in a ratio of %32 for B-16VpHM6mIL-2, %51.7 for B-16VpHM6mLys and %49.2 for B-16VpHM6mLys + B-16VpHM6mIL-2. There was notumor development in the mice living after experiment 3 treatments when B-16cells were re-injected (experiment 4) indicated that a protective immunity wasarisen. These results had showed that, lysozyme can also be used in the tumorimmunotherapy if it be secreted in tumor cells in a high level through an agentsuch as plasmid DNA.Key Words: B-16V, C57BL/6, DNA Vaccine, IL-2, Lysozyme, pHM6 plasmid vector,Tumor immunotherapy