Combined IL-2 And IL-12 Gene Therapy For Murine Hepatocellular Carcinoma

ObjectiveIn recent years, the advance of HCC immunogene therapies has been restricted by low therapeutic efficacy of solo gene and inefficiency of transferring gene into target cells due to devoid of ideal vectors, so combined genes therapy would be a promising alternative. Great deals of researches have indicated that interleukin 2 and interleukin 12 genes would be potentially therapeutic genes, which bring into full play by activating natural killer cell (NK) and cytotoxic T lymphocyte (CTL). To explore gene expression, immune enhancement and antitumor efficacy of intra-tumoral administration of eukaryotic plasmid DNA encoding both mIL12 and hIL2, compared with plasmid DNA expressing mIL12 or hIL2, a randomized controlled trial was used in murine models with the subcutaneously grafted H22 hepatocellular carcinoma. Based on those results, researches will be further performed on HCC immunogene therapy with combined IL-2 and IL-12 gene through efficient gene transfer technologies.Methods1. Plasmid vectors encoding hIL2-mIL12, mIL12 and hIL2 were constructed with molecular cloning methods.2. After plasmid DNA transfecting cell COS-7 with liposome, the expression of the corresponding cytokine was examined in eukaryotic cell respectively through Enzyme-linked immunosorbent assay (ELISA). 3. The proliferation assay of T lymphoblasts was performed for measuring the biological activity of the expressed mIL12 and the proliferation assay of thymocytes for detecting the biological activity of the produced hIL2.4. After intra-tumoral administration of plasmid DNA 20μg, the expressed cytokines were detected in the serum at different time through ELISA in the pDC511hIL2-mIL12, pDC511hIL-2, pDC511mIL-12 and pDC511 group respectively.5. Following more injection of naked plasmid DNA 20μg, mean diameter of the tumor mass, survival time and livability were measured in each murine model group. 6. Lactic dehydrogenase (LDH) assay was used to examine whether or not treatment with different plasmid DNA could induce systemic cytolytic activity of lymphocytes against parental H22 cells.7. Histopathological analysis was operated before and after administration of plasmid DNA vectors in each murine model group.8. Statistical analysis: All graphs were depicted with software EXCEL.Differences in tumor growth were statistically analyzed using the repeated measures ANOVA test. Statistical evaluations of survival time were performed using PL test. Differences in livability were statistically analyzed using Fisher'exact probability test.Results1. Plasmid vectors pDC511hIL2-mIL12, pDC511hIL-2 and pDC511mIL-12 were constructed successfully.2. All plasmid vectors could express corresponding cytokine in eukaryotic cell. As measured by ELISA after each DNA vector transfecting cells COS-7, the supernate in the pDC511hIL2-mIL12 group contained 3087.18pg/(ml·24h·106)cell hIL2 and 674.56pg/(ml·24h·106)cell mIL12, 504.37pg/(ml·24h·106)cell mIL12 in the pDC511mIL12 group and 4274.19pg/(ml·24h·106)cell hIL2 in the pDC511hIL2 group. No cytokine was detected in the pDC511 group.3. After plasmid DNA transfecting cell COS-7, the supernate collected from the pDC511hIL2-mIL12 group had more capacity of stimulating the proliferation of T lymphoblasts or thymocytes than from the pDC511mIL12 group or pDC511hIL2 group.4. HIL2 and mIL12 expression in the serum rose to a peak at 1-7 days and 1-10 days post injection of plasmid pDC511hIL2-mIL12 DNA, ranging from 30.22±5.25pg/ml to 140.85±8.88pg/ml and from 1.01±1.76pg/ml to 75.52±17.19pg/ml respectively. HIL2 expression rose to a peak at 2-7 days after injection of plasmid pDC511hIL2 DNA ranging from 30.24±1.23pg/ml to 98.34±6.94pg/ml, and mIL12 at 2-10 days post injection of plasmid pDC511mIL12 DNA ranging from 0.69±0.60pg/ml to 49.38±3.63pg/ml. The hIL2 or mIL12 expressed in the pDC511hIL2-mIL12 group had statistically difference compared with the pDC511hIL2 (p<0.01) or pDC511mIL12 (p=0.04) in the serum at different time.Growth of li...