| Background and objectiveWith the further study of liver cancer, the role of liver microenvironment inhepatocellular carcinoma development has gradually gained extensive attention. Although ithas been confirmed that the combination of IL-2and IL-12can get a obvious anti-tumoreffect, the study about tumor microenvironment after the combination treatment of IL-2andIL-12has not been reported yet.In our study, inducible plasmids encoding IL-2and IL-12were injected into mice byhydrodynamic injection methods into the tail vein. We intended to observe the inhibition ordelay of tumor growth after the change of microenvironment in liver or spleen.MethodsPlasmids encoding mIL-2and mIL-12contained RU486regulated system and theliver-specific promoters. Orthotopic hepatocellular carcinoma model was established in mice.Three experimental groups included IL-2, IL-12and the combined group, and two controlgroups included LacZ and Saline group. ELISA was employed to test the expression level ofIL-2and IL-12in serum. Portion of mice were sacrificed at the end of the inducement, andtumor size was measured. Stable tissue lymphocytes were separated through enzymedigestion and Percoll density gradient. We used flow cytometry to analyze immunephenotype and distribution of cells in liver tumors and spleen. HE and immunohistochemicalstaining were employed to observe histological changes and tumor PCNA and VEGFexpression separately. Lifetimes of remaining mice were observed.Results1. Tumor growth and mouse survivalThrough the measurement of liver tumor size, the average volume of tumors in eachgroup was555.96±122.36mm3(Saline),554.63±133.08(LacZ) mm3,276.68±16.46mm3(IL-2),148.32±28.17mm3(IL-12) and60.59±27.06mm3(IL-2/IL-12) separately. Tumor sizein three experimental groups was smaller than control groups, especially in combined group,with the tumor size significantly smaller than the control group and even smaller than the single dose of IL-2or IL-12group.The mean survival times of mice in each group was24.75±0.86d (Saline),24.89±0.98d (LacZ),25.00±1.74d (IL-2),38.00±2.94d (IL-12) and40.11±6.09d (IL-2/IL-12).Although there is no statistical difference between the IL-2and control groups, but in IL-12and IL-2/IL-12group was prolonged than the control groups.2. Morphological features of liver and tumorsHE staining of liver revealed that tumors in control groups were large. Although thereare necrosis tissues in some large tumors, the boundaries between tumor and normal tissuewere unclear, tumor cells showed invasive growth, and rare amounts of lymphocytesinfiltrated in the portal area. However, tumors in IL-2group, IL-12group and combinedgroup confined to the edge of hepatic lobes and showed focal distribution. The boundariesbetween tumor tissue and normal tissue were clear, and the complete morphology of tumorcells was rarely found. Especially in combined group, there was large area of necrosis in thetumor tissue, and some tumor cells disappeared and infiltration by mononuclear cell instead.Immunohistochemical staining showed that the high PCNA and VEGF positive rate inthe tumors of mice treated with in control groups, and most of the positive cells were darkbrown. But the low positive rate in three experimental groups, and most of the positive cellswere light brown. There was significant difference among these groups. Positive cell rate ofthe combined group is less than the single dose IL-2and IL-12group, and there is nodifference between IL-2and IL-12group.3. The expression of cytokines in the serumThe expression of mIL-2was seen in IL-2group and combined group. The expressionof mIL-2in IL-2group was higher than combined group, in which expression of IL-2wasonly58.65%of the IL-2group. The mIL-12was detected in IL-12and combined group. Theexpression of IL-2in combined group was higher than IL-2group, in which expression ofIL-12was only76.38%of the combined group.4. The changes of liver and splenic local tumor microenvironmentThe ratio of CD4+/CD8+in liver tumors declined in three experimental groups thancontrol group, this ratio in combined group declined significantly compared with single doseIL-2and IL-12group. The ratio of CD8+/Treg in three experimental groups was higher thancontrol group, especially in combined group this ratio surpass single dose IL-2and IL-12group. Nevertheless, the ratio of CD4+/CD8+only combined group declined in spleen. Conclusions1. The regulatory interleukin2in combination with interleukin12could inhibition tumorcell proliferation and VEGF expression, thereby delayed tumor growth.2. The regulatory interleukin2in combination with interleukin12could reduced the ratioof CD4+/CD8+and increase the ratio of CD8+/Treg in liver tumor, improved local tumormicroenvironment. |
PDF Full Text Request