Study Of IL-24Gene Therapy For Murine Melanoma

Objective:To study of recombinant plasmid pEGFP-N1-IL-24gene expression in murine melanoma Xenografts and its effect on inhibition of melanoma.Methods:1. B16cells which frozen in liquid nitrogen were cultured and passaged in RPMI-1640supplemented with10%FBS at37℃under5%CO2.2. All C57BL/6mice were acclimatized to the laboratory environment for one week before the experiment.Then collect logarithmic phase B16cells under sterile conditions, each with5×106cells/ml inoculated back subcutaneous in mice, the transplanted tumors were observed every day.3. After established the model of melanoma Xenografts in C57BL/6mice, the18mice were randomly divided into the following3groups:IL-24recombinant plasmid as the experimental group、empty vector plasmid group and PBS group as control groups.Each group contained6mice.Then xengraft tumors in IL-24recombinant plasmid group was injected with lipofectin encapsulated pEGFP-N1-IL-24(10μg) every3days,at the same time, lipofectin encapsulated pEGFP-N1(10μg) and PBS(0.2ml) as empty vector control and negative control,a total of four injections.Before each injection of drugs with a vernier caliper to measure tumor length (L) and short diameter (W), then calculate the tumor volume and draw the tumor growth curve.4. On the3day after the last injection the drugs,the mice were sacrificed and tumor tissues were dissected to frozen.5. The expression of IL-24mRNA was detected by RT-PCR in three groups of transplanted tumors.6. Western blot was used to determine the expressions of Bax in all groups of tumor tissues.Results1. After18mice were inoculated subcutaneously with melanoma B16cells,each mouse subcutaneous tumor diameter covered5mm after12days. Melanoma xenograft mouse model was successfully constructed.2. The tumor growth curve shows that the volume of IL-24recombinant plasmid group transplanted tumor significantly less than the empty vector plasmid group and PBS group, the difference was statistically significant (P<0.05).But there was no significant difference between the empty vector plasmid group and the PBS group.(P>0.05)3. All tumor tissue were extracted RNA and determined by RT-PCR, IL-24recombinant plasmid group showed611bp position has a specific band,while the empty vector group and PBS group has not occured the expected bands.4. The Bax proteins were extracted in each group transplanted tumor tissue by Western blotting confirmed the proapoptotic protein Bax were expressed in recombinant plasmid pEGFP-N1-IL-24group, while the empty vector plasmid group and PBS group,neither of the Bax protein expressed.Conclusion1. IL-24gene stable express the protein in melanoma tissue.2. IL-24gene recombinant plasmid inhibited the growth of transplanted tumors of melanoma cells in mice.3. IL-24gene has promoted the Bax protein activation function on melanoma tissue.