| Both interleukin 2 (IL-2) and interleukin 12 (IL-12) are potent cytokines with robust antitumor effects that play a crucial role in antitumor immunity. Because of the complementarity of the antitumor mechanism, IL-2 combined with IL-12 has stronger anti-tumor effect than single one, and effectively reduce the toxicity.The recombinant proteins of IL-2 and IL-12 had powerful antitumor activities in clinical treatment, but the toxicity limited the clinical application, thus gene therapy has been investigated as a new means to decrease or eliminate the toxicity. Gene transfer mediated by viral vectors is highly effective and results in the retardation of tumor growth or eradication. But the production of viral vectors is complicated. Moreover, the viral vectors may induce the antiviral immunity. The naked DNA of non-viral vectors is widely concerned because of its advantages including safety and simplicity. However, the low transfection efficiency and susceptible to degradation limit the application. High expression of transgene in vivo could be achieved by hydrodynamic injection of naked DNA.In this study, plasmid expressing IL-2 gene was constructed and combined with IL-12 to explore the antitumor activity and side effects of IL-2 and IL-12 gene.MethodsAfter construction and confirmation with restriction enzymes, the plasmid pCMV-mIL-2 was transfected into the cells in vitro, or injected into the mice by hydrodynamic injection. The expression of mIL-2 in supernatant of cultured cells and mIL-2 & IFN-γin serum of mice was determined by ELISA. DNA and RNA were extracted from major organs of mice for PCR or RT-PCR.H22 hepatoma cells together with pCMV-mIL-2 or/ and pCMV-mIL-12 plasmids were injected into mice by hydrodynamic injection. The blood was taken at indicated time points. The expression of mIL-2, mIL-12 and IFN-γin serum was detected by ELISA. Some of the mice were sacrificed 7 days after injection to record the weight of the organs and tumor nodules of the organs. HE staining was applied to analyse the morphological changes of the liver, spleen and lungs. The other mice were kept to monitor the survival time. Results1. Structure and functional identification of plasmid pCMV-mIL-2The expected fragments were obtained after digestion of pCMV-mIL-2 by Hind III restriction enzymes, which confirmed that the structure of cloned plasmid was correct. High level of mIL-2 was detected in supernatant of cultured cells deriving from different tissues after transfection with pCMV-mIL-2.2. Expression of mIL-2 and IFN-γin vivoDifferent doses of pCMV-mIL-2 were injected into the mice by hydrodynamic injection. Blood serum was collected at different time points to meseaure mIL-2 and IFN-γexpression. The kinetic expression pattern was similar although the plasmid doses were various. MIL-2 level increased sharply in 4~8 h after injection, decreased abruptly at 24 h, and mIL-2 was undetectable on day 3. IFN-γreached a maximal level on day 7.The level of mIL-2 and IFN-γwas increased with the dose of pCMV-mIL-2.Twoμg pCMV-mIL-2 was given to mice. Repeated injection of pCMV-mIL-2 with an interval of 7 days was performed after initial administration. MIL-2 level at a concentration of 4629.46±1795.85 pg/ml after the second injection was close to the first one. The third injection induced the highest level of mIL-2 (12322.54±983.12 pg/ml), which was 3.65-fold higher than that of the first one, 2.66-fold higher than that of the second one. The fourth injection produced similar level of mIL-2 (12016.74±1065.85 pg/ml) as the third one.3. Morphological changes of the liver after pCMV-mIL-2 injectionTwoμg pCMV-mIL-2 was injected into the mice, the livers were collected at different time points for HE staining. The loosen cytoplasm and necrosis of hepatocytes appeared at 4 ~ 8 h after injection. The necrosis disappeared and the infiltration foci of mononuclear cells appeared on day 3. The number and area of mononuclear cells foci reduced on day 7.Repeated injection of pCMV-mIL-2 with an interval of 7 days was performed after initial administration. The mice were sacrificed on day 7 after each injection for morphological evaluation of the liver. Increased infiltration foci were found in portal areas after the third and fourth injection. Monocytes, lymphocytes and few neutrophils were present in the foci.4. Distribution of pCMV-mIL-2 in vivoThe mice were sacrificed at different time points respectively after 10μg pCMV-mIL-2 was injected into mice. The liver, spleen, lungs and kidneys were taken to extract RNA and DNA. PCR results showed that pCMV-mIL-2 plasmid was transferred to the main organs of mice successfully, which was mainly in the liver. RT-PCR demonstrated that transcription of transgene was correctly run.5. Combined IL-2 and IL-12-encoding plasmids suppress tumor formation in miceSome of mice were sacrificed on day 7 after injecting H22 cells and plasmid. Mean number of tumor nodules in the liver was 2.80±6.26 in saline group, 3.00±5.10 in pCMVβgroup and 0.33±0.82 in pCMV-mIL-2. Mean number of tumor nodules in the lungs was 7.80±7.34 in saline group, 3.20±3.11 in pCMVβgroup and 0.17±0.41 in pCMV-mIL-2 group. No tumor nodules were found in pCMV-mIL-12 and IL-2/IL-12 group.In dead mice, mean number of tumor nodules in the liver was 19.29±21.91 in saline group, 15.67±5.72 in pCMVβand 2.50±3.39 in pCMV-mIL-2 group. Mean number of tumor nodules in the lungs was 36.43±26.69 in saline group, 9.67±8.82 in pCMVβgroup and 2.83±4.40 in pCMV-mIL-2 group. No tumor nodules were found in group pCMV-mIL-12 and IL-2/IL-12 until day 84. The survival time in group pCMV-mIL-12 and IL-2/IL-12 was prolonged than others.6. Cytokine expression after injection of combined IL-2 and IL-12-encoding plasmidsAt 8 h after injection, high level mIL-2 was detected in pCMV-mIL-2 and IL-2/IL-12 group, serum mIL-2 level was the highest in pCMV-mIL-2 group, and the level in IL-2/IL-12 group was 70.3% of pCMV-mIL-2 group. High level mIL-12 was detected in pCMV-mIL-12 and IL-2/IL-12 group, mIL-12 level was the highest in pCMV-mIL-12 group, and the level in IL-2/IL-12 group was 50% of pCMV-mIL-12 group. At day 3, IFN-γwas detected in pCMV-mIL-12 and IL-2/IL-12 group, the level in pCMV-mIL-12 group was the highest, and in IL-2/IL-12 group was 13.98% of pCMV-mIL-12 group. At day 7 after injection, IFN-γwas detected in pCMV-mIL-2, pCMV-mIL-12 and IL-2/IL-12 group, the level in pCMV-mIL-12 group was the highest, in IL-2/IL-12 group was 40% of pCMV-mIL-2 group and 18.76% of pCMV-mIL-12 group.7. Effects on spleen, liver and lungs after injection of IL-2 and IL-12-encoding plasmidsSome mice were sacrificed at day 7 after injected H22 cells and plasmids. The weight of the spleen in group pCMV-mIL-12 was the heaviest, it was 69.0% of pCMV-mIL-12 group in IL-2/IL-12 group. The number of giant cells in pCMV-mIL-12 group was the most, it was 56.0% of pCMV-mIL-12 group in IL-2/IL-12 group. More necrotic foci were found in the liver of pCMV-mIL-12 group. The weight of the lungs was the heaviest and the alveolar septa were the thickest in pCMV-mIL-2 group. Conclusion1. The plasmid expressing mIL-2 gene is successfully constructed, which can be highly expressed in mice.2. Combination with IL-2 and IL-12 gene can efficiently suppress H22 tumor formation in mice and prolong the survival period.3. Combination with IL-2 and IL-12 gene can reduce the side effects, enhance the antitumor activity. The anti-tumor mechanism relates to the expression of IL-2, IL-12, IFN-γ, and inflammatory cells infiltration. |
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