The Effect Of Recombinant Adenovirus-mediated IL-23 On Human Hepatocellur Cacinoma Cells HepG2

Objective: Liver cancer is one of the most common malignancy. Currently,the effects of surgery, chemotherapy, radiation are not satisfactory, and it is also a serious threat to the survival of people's health.It is urgently needed to explore a new treatment methods. In recent years,the immune and gene therapy for the liver cancer have opened up a new path.The way cytokines based to treat cancer has gradually become important.It has been proved that interleukin-12(IL-12) can inhibit primary and metastatic liver tumor in mice. As one of family members of IL-12,interleukin-23 (IL-23),the latest discovered cytokine to colon cancer,melanoma,breast cancer, esophageal cancer,pancreatic cancer,have a strongly inhibitory effect.The result is encouraging.It provides a more secure way to clinical for the toxic is lower than IL-12. At present, IL-23 on liver cancer in animals has also been made more satisfactory efficacy. However, it remains unclear whether or not to have a direct toxic on liver cancer cells and whether or not to related to cell apoptosis ..The assay is to investigate the mechanism of IL-23 on liver cancer by exploring the influence of recombinant adenovirus-mediated IL-23 on human hepatocellular carcinoma cells hepG2.It will provide a theoretical basis for the treatment of liver cancer.Methods: The recombinant adenovirus (adv-EGFP-IL-23) modified with human interleukin-23 and enhanced green fluorescent protein gene were infected hepG2 cells at an plural (MOI) 10,50,100,200,500, respectively, as well as non-transfected hepG2 cells for control group.After 24 hours, the cells were observed under fluorescence and inverted microscopy, repectively, transfection efficiency was measured by flow cytometry and determine the best MOI. The expression of IL-23 (p19 and p40 subunits) in hepG2 cells were determined by RT-PCR after the transfection of adv-EGFP-IL-23. The growth inhibition of control group, adv-EGFP group, adv-EGFP-IL-23 transfection group were measured by MTT assay at different times (0,24,48,72 hours) . cell morphology was observed in each group at different times. After PI staining, the rate of cell apoptosis was measured by flow cytometry after 24,48 hours. Statistical treatment: The experimental data was analyzed by One-Way ANOVA.Results: adv-EGFP-IL-23 can be highly efficient transfected into hepG2 cell. when the MOI of 100, the transfection efficiency was up to 91.63±4.83%.,and remarkably higher than those in groups of MOI of 10,50, P <0.05 .when compared with groups MOI of 200,500,there was no significantly different, P > 0.05. so to identify 100 as the best MOI. In the hepG2 cells transfected with adv-EGFP-IL-23, the message RNA expression of IL-23 (p19 and p40) could been discovered. The growth curve of the three groups were a good coincidence, was no significant difference, P > 0.05,indicated IL-23 can not inhibited hepG2 cells. Flow cytometry analysis show that the rate of cells apoptosis of different groups at different times (24,48 hours) , was no significant difference, P > 0.05.Conclusion: The recombinant adenovirus carrying interleukin-23 gene can be highly efficient transfected into hepG2 cells and expressing IL-23 (p19 and p40) mRNA in cells. The growth rate and apoptosis rate of three group hepG2 cells were no significant difference , indicate that IL-23 itself may has no direct toxic effects and can not inhibited hepG2 cells, which in accordance with reported in the literature, but also an indirect proof of IL-23 anti-tumor effect is mediated by the immune cells, depend on the activation of TH1 response and CTL function.