Experimental Study Of Polygene Therapy On Murine Melanoma

Objective:To study the inhibitory activity and mechanism of recombinant plasmid pEGFP-N1-IL-24gene and pGEG-IL-12gene single application and combined application to melanoma B16cells in mice xenografts.Methods:The B16cells were recoveried and cultured, then inoculated on the back of the6-week old C57BL/6mice, to establish the melanoma model of B16cells. The results showed that there was a tumor with the diameter of0.05cm on the back of mice about12days after inoculation. Then the tumor-bearing mouse were randomly divided into5groups,8mouse per group,and they were respectively injected the recombinant plasmid liposome of pEGFP-N1-IL-24(IL-24group), pEGFP-N1-IL-24,pGEG-IL-12(IL-24+IL-12group), pGEG-IL-12(IL-12group) and pEGFP-N1(empty plasmid group) and PBS buffer solution (PBS group) into the tumors. Before injection, the long and short diameter of the tumor were measured, and calculate the volume, draw the growth curve of the tumor. The histology and morphology of the tumor tissue was observed by HE staining. The mRNA of IL-24, IL-12, Bax, Bcl-2and VEGF were detected in tumor tissue by RT-PCR, The Protein expression of Bax, Bcl-2, VEGF, caspase-3and caspase-12in tumor tissue were detected by Western blot.The data were analyzed with SPSS19.0and the results were expressed by x±s.Results:1. The growth curves showed that the volume of the tumor in the IL-24group, IL-12group was significantly smaller than that in the empty plasmid group, PBS group (P<0.05), and the volum of tumor in the IL-24+IL-12was smallest(P<0.05).2.7days after the last injection of the gene, the mice were sacrificed and taken the tumor tissue. HE staining showed that the growth, morphology, structure of tumor cell was well, and melanin formation in the cytoplasm, enlarged and hyper-chromatic nuclei and other typical pathological features of malignant melanoma were visible in empty plasmid group and PBS group; while most cell growed badly, and can be observed that irregular shape, cell shrinkage, nuclear dissolution, sparse, karyolysis, deformation, patches of necrosis in the recombinant plasmid group.3. After RT-PCR experiments, the results showed that there was the mRNA of IL-24, IL-12in the IL-24group and IL-12group respectively, and both of them were detected in the the IL-24and IL-12group, while it is opposite in the empty plasmid group and PBS group; and the mRNA level of Bax in the IL-24group and IL-12group is higher than that in the empty plasmid group and PBS group (P<0.05), with the highest in the the IL-24and IL-12group, in contrast, the mRNA level of Bcl-2and VEGF were on the contrary (P<0.05).4. After Western Blot experiments, the Protein expression level of Bax,caspase-3and caspase-12were discovered in the IL-24group and IL-12group was significantly higher than that in the empty plasmid group and PBS group (P<0.05), and the highest in the the IL-24and IL-12group, but the Protein expression level of Bcl-2and VEGF were on the contrary (P<0.05).Conclusion:1. The tumor-bearing mouse model of melanoma was established successfully by inoculating B16cells on the back of mice.2. IL-24and IL-12has inhibitory effect to the mice B16melanoma growth, and the effect of the united application is stronger.