Abstract: Lu Lan, Xie Conghua, Zhang Haozhong, Xu Lyuye, Shi Xingwei, Xie Jun, Che Biao, Ding Wen Department of Radiation and Chemotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China （Lu L, Xie CH）; Department of Internal Medicine, General Hospital of the Yangtze River Shipping, Wuhan 430010, China （Zhang HZ, Xu LY, Shi XW, Ding W）; Department of Surgery, General Hospital of the Yangtze River Shipping, Wuhan 430010, China （Xie J, Che B） Corresponding author: Xie Conghua, Email: chxie_65@whu.edu.cn 【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?N1?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 . 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 B16 cells. After effector cells were co?cultured 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?transfected 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.