IL-15上调NKG2D表达对CIK细胞杀伤活性的增强效应

Enhancement of Cytotoxicity against IL-15 Up-regulate NKG2D Expression on Cytokine-induced Killer Cells

目的观察IL-15对细胞因子诱导的杀伤细胞(Cytokine-induced killer cells,CIK)NKG2D受体表达及其对食管癌EC9706细胞杀伤活性的影响。方法体外分离外周血单个核细胞,分为两组。对照组:干扰素-γ、白细胞介素-2、CD3单抗诱导培养CIK细胞。IL-15组:加用IL-15培养。流式细胞仪检测细胞免疫表型及CD3+细胞、CD56+细胞表面NKG2D的表达,LDH法测定第14天细胞在效靶比20∶1、30∶1时对EC9706细胞的杀伤活性;效靶比30∶1时,观察NKG2D单抗封闭细胞表面NKG2D分子后对两组细胞杀伤活性的影响。结果随着培养时间的延长,CIK群体细胞及CD56+细胞表面NKG2D表达逐渐增强,IL-15组与对照组相比差异有统计学意义(P<0.05);效靶比20∶1、30∶1时,IL-15组细胞对EC9706细胞的杀伤活性均较对照组明显增强,差异均有统计学意义(P<0.05);效靶比30∶1时NKG2D单抗封闭CIK细胞表面NKG2D分子后,对照组细胞、IL-15组细胞对EC9706细胞的杀伤活性均较阻断前明显下降,差异均有统计学意义(P<0.05)。结论 IL-15上调CIK细胞表面NKG2D分子表达,增强CIK细胞对EC9706细胞的杀伤活性,CIK细胞通过NKG2D发挥作用。

Objective To analyse the effects of IL-15 on the expression of NKG2D and the cytotoxicity of cytokine-induced killer（CIK） cells against human esophagus carcinoma cell EC9706 in vitro.Methods Peripheral blood mononuclear cells were isolated from healthy donors then divided into two groups： the control group（cells were cultured in the presence of IFN-γ,anti-CD3 antibody and IL-2） and IL-15 group（cells were cultured in the presence of IFN-γ,anti-CD3 antibody,IL-2 and 10 ng/ml IL-15）.Phenotypic characteristics of CIK cells and the expression of NKG2D were analyzed by flow cytometry.After 14 days culture,cytotoxicity of CIK cells against EC9706 cells was measured by using a standard LDH releasing assay.Effector cells were added to target cells at E∶T radios of 20∶1,30∶1.In blocking experiments,NKG2D monoclonal antibody was added to CIK cells for 15 min before plating at 30∶1 E∶T ratio.Results NKG2D molecules were significantly up-regulated during the culture period on CD3＋ cells（the CIK cells population） and the CD56＋cells.The differences of NKG2D expressions were significant between IL-15 group and the control group（P0.05）.Cytotoxicity of CIK cells treated by the IL-15 group was higher than that of the control group,both at 20∶1 E∶T ratios and at 30∶1 E∶T ratios（P0.05）.When the NKG2D molecules on CIK cell ular membrance were blocked by the NKG2D monoclonal antibody,the cytolytic activity in the control group cells and IL-15 group cells was significantly inhibited.Conclusion IL-15 up-regulated the expression of NKG2D on CIK cells,which enhanced the NKG2D mediated cytotoxicity against EC9706 cells.CIK cells played a role through the NKG2D molecules.