目的：探讨IL-12对细胞因子诱导的杀伤 (cytokine-induced killer，CIK) 细胞的表型和CIK细胞在体外对食管癌EC9706细胞杀伤活性的影响。 方法: 体外分离人外周血单个核细胞，分为两组：对照组以IFN-γ、IL-2和CD3单抗诱导培养CIK细胞；IL-12组在对照组基础上加用IL-12培养。培养至第14天，流式细胞仪检测两组CIK细胞的免疫表型；LDH释放法测定效靶比20 ∶1、30 ∶1时两组CIK细胞对EC9706细胞的杀伤活性；观察效靶比30 ∶1时，NKG2D单抗对两组CIK细胞杀伤活性的影响。 结果: IL-12组与对照组相比较，CIK细胞免疫表型的CD3+CD56+细胞比例\[(28.23±1.71)% vs (16.34±059) %, P<0.05\]、CD3+细胞及CD3+CD56+细胞中NKG2D的表达\[(77.45 ±2.15)% vs (66.87±0.73)%, (92.94±077)% vs (82.18±066)%；均P<0.05\]、CD3+细胞中穿孔素的表达\[(51.78±0.63)% vs (43.54±0.95)%，P<0.05\]都明显增强；CD3+细胞颗粒酶B表达无明显变化\[(26.90±0.67)% vs (26.76±0.33)%，P>0.05)\]。效靶比20 ∶1和30 ∶1时，IL-12组CIK细胞对EC9706细胞的杀伤活性均较对照组明显增强\[(43.92±1.67)% vs (35.34±1.22)%，(55.95±0.88)% vs (43.91±110)%；均P<005\]；以NKG2D单抗阻断后，IL-12组、对照组CIK细胞杀伤活性均明显下降\[(19.72±0.56)% vs (55.95±0.88)%, (19.83 ±1.20)% vs (43.91±1.10)%;均P<0.05\]。 结论: IL-12能够上调CIK细胞NKG2D和穿孔素的表达，从而增强CIK细胞对EC9706细胞的杀伤活性。

Objective: To explore the effects of IL-12 on the phenotypes of cytokine-induced killer (CIK) cells and the cytolytic activity of CIK cells against human esophageal carcinoma EC9706 cells in vitro. Methods: Peripheral blood mononuclear cells were isolated from healthy donors. Two groups were designed: a control group (cells were cultured in the presence of IFN-γ, IL-2 and anti-CD3 antibody) and IL-12 group (cells were cultured in the presence of IFN-γ, anti-CD3 antibody , IL-2 and IL-12 ). After 14-day culture, the phenotypes of CIK cells in the control and IL-12 groups were analyzed by flow cytometry. The cytotoxic activity of CIK cells on EC9706 cells was measured by LDH releasing assay at effect-to-target (E ∶T) cell ratios of 20 ∶1, 30 ∶1. The anti-NKG2D monoclonal antibody was added to CIK cells to detect its effect on the cytotoxic activity of CIK cells at E ∶T ratio of 30 ∶1. Results: In comparison to the control group, the proportion of CD3+CD56+cells, NKG2D expressions on CD3+ and CD3+CD56+cells, and perforin expression in CD3+ cells were higher in the IL-12 group (\[28.23±1.71\]% vs \[16.34±0.59\]%; \[77.45±2.15\]% vs \[66.87±0.73\]%,\[92.94±0.77\]% vs \[82.18±0.66\]%; \[51.78±0.63\]% vs \[43.54±095\]%; all P<0.05). Whereas, no significant change was observed in the granzyme B expression in the CD3+ cells \[(26.90±0.67）% vs (26.76±033）%，P>0.05\]. The cytolytic activity of CIK cells against EC9706 cells was increased significantly in the IL-12 group (E ∶T ratio of 20 ∶1, \[43.92± 1.67\]% vs \[35.34±1.22\]% ; E ∶T ratio of 30 ∶1, \[55.95±0.88\]% vs \[43.91±1.10\]%, all P<0.05). The cytotoxicity of CIK cells in the IL-12 and the control groups were significantly inhibited by anti-NKG2D monoclonal antibody (\[19.72±0.56\]% vs \[55.95±0.88\]%, \[19.83±1.20\]% vs \[43.91±1.10\]%, all P<0.05). Conclusion: IL-12 up-regulates the NKG2D and perforin expressions on CIK cells, enhancing their cytotoxicity against EC9706 cells.

河南省科技计划资助项目（No. 201143）