ABSTRACT
Objective To evaluate cytotoxic effects of cytokine-induced killer cells (CIK cells) transfected with the interleukin-2 (IL-2) gene on malignant melanoma cells.Methods Mouse spleen cells were extracted,lymphocyte cells were separated,and CIK cells were prepared from these lymphocyte cells.PEGF-N1 plasmids containing IL-2 gene (PEGF-NI-IL-2) were transfected into CIK cells.Fluorescence microscopy was used to observe transfection products,and reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to determine the IL-2 mRNA expression.Then,effector cells such as CIK cells and IL-2-transfected CIK cells were separately co-cultured with target cells (B16 melanoma cells) at effector-target ratios of 10∶ 1,20∶1 and 40∶1,then 4-hour lactate dehydrogenase release assay was performed to evaluate cytotoxic effects of the two kinds of CIK cells on B 16 cells.After effector cells were cocultured with target cells at the effector-target ratio of 40∶1 for 48 hours,enzyme-linked immunosorbent assay (ELISA) was conducted to detect levels of IL-2,interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatant of the two kinds of CIK cells.Finally,mouse models of melanoma were established,and a total of 28 melanoma-bearing mice were divided into 4 groups to be peritumorally injected with 0.2 ml sodium chloride physiological solution (control group),100 IU IL-2 solution (IL-2 group),CIK cell suspension at a cell density of 1 × 106 cells per milliliter (CIK group) and IL-2-transfected CIK cell suspension at a cell density of 1 × 106 cells per milliliter (IL-2-transfected CIK group) respectively.Tumor morphology,tumor inhibition rate and cell apoptosis rate were used to evaluate tumor growth in the above groups.If data were normally distributed,t-test was used for comparing means between two groups,and analysis of variance and least significant difference (LSD)-t test were used for comparing means among multiple groups.Results Fluorescence microscopy and RT-PCR both showed that IL-2 was successfully transfected into CIK cells.The cytotoxic effect of IL-2-transfected CIK cells on B16 cells was strongest at the effector-target ratio of 40:1.Levels of IL-2,IFN-γ and TNF-α were also significantly higher in the supernatant of IL-2-transfected CIK cells [(1107.26 ± 6.49) pg/ml,(50.01 ± 3.35) pg/ml,(39.86 ± 3.25) pg/ml] than those in that of CIK cells [(51.09 ± 3.85) pg/ml,(32.71 ± 2.43) pg/ml,(30.11 ± 3.08) pg/ml,t =442.60,14.93,6.89,all P ＜ 0.01].Animal experiments showed that the tumor volume obviously increased in the control group (P ＜ 0.05),but significantly decreased in the IL-2 group,CIK group and IL-2-transfected CIK group (all P ＜ 0.001) after intervention compared with those before intervention.Furthermore,the tumor volume in the IL-2-transfected CIK group was significantly less than that in the other three groups (all P ＜ 0.01),but no significant difference was observed between the IL-2 group and CIK group (P ＞ 0.05).In addition,the apoptosis rate was significantly higher in the IL-2 group,CIK group,and IL-2-transfeeted CIK group than that in the control group (all P ＜ 0.01).The apoptosis rate and tumor inhibition rate were significantly higher in the IL-2-transfected CIK group than those in the IL-2 group and CIK group (all P ＜ 0.01),but insignificantly different between the IL-2 group and CIK group (P ＞ 0.05).Conclusion IL-2-transfected CIK cells had stronger killing effects on malignant melanoma.