NKG2D介导CIK细胞对食管癌EC9706细胞杀伤作用的体外研究

NKG2D Mediated Cytotoxicity of Cytokine-Induced Killer Cells Against Human Esophageal Carcinoma Cell Line EC9706 in Vitro

目的:研究细胞因子诱导的杀伤细胞(Cytokine-induced killer cells,CIK)对食管癌EC9706细胞株的体外杀伤活性。方法:流式细胞仪检测EC9706细胞NKG2D配体的表达;体外分离健康者外周血单个核细胞,干扰素-γ、白细胞介素-2、CD3单抗诱导培养,流式细胞仪检测0、14天的细胞CD3^+CD56^+、NKG2D的表达,乳酸脱氢酶释放法测定培养14天的CIK细胞在效靶比10:1、20:1、30:1、40:1、50:1时对EC9706细胞的杀伤活性;效靶比30:1时,观察NKG2D单抗封闭CIK细胞表面NKG2D分子后对CIK细胞杀伤活性的影响。结果:EC9706细胞表达MICA、ULBP2,不表达MICB、ULBP1、ULBP3。0、14天细胞表面NKG2D表达率分别为(26_30±1.12)%、(67.13±1.34)%,差异有统计学意义(P<0.05);CD3^+CD56^+细胞分别为(0.68±0.07)%,(56.55±2.01)%,差异有统计学意义(P<0.05);效靶比10:1、20:1、30:1、40:1、50:1时CIK细胞对EC9706细胞的杀伤活性分别为(28.81±0.47)%、(37.78±0.22)%、(44.31±1.06)%、(47.25±0.47)%、(57.62±0.94)%;随着效靶比增高,CIK细胞对EC9706细胞的杀伤活性明显增强(P<0.05);效靶比30:1时NKG2D单抗封闭CIK细胞表面NKG2D分子后,CIK细胞对EC9706细胞的杀伤活性为(30.29±1.45)%,与阻断前(44.31±1.06)%相比差异有统计学意义(P<0.05)。结论:体外CIK细胞培养增殖过程中,NKG2D分子表达逐渐上调,CD3^+CD56^+细胞逐渐增多,CIK细胞杀伤EC9706细胞通过NKG2D发挥作用。

Objective： To investigate the cytotoxicity of cytokine-induced killer （CIK） cells against human Esophageal Carcinoma cell line EC9706, in vitro. Methods： The expression of NKG2D ligands of EC9706 cells were analyzed by flow cytometry. CIK cells were generated from peripheral blood mononuclear cells of healthy donors by culturing cells in the presence of IFN-γ, anti-CD3 antibody and IL-2, in vitro. The percentage of CD3＋ CD56＋ and the expression of NKG2D in the bulk CIK cells at days 0 and 14 were analyzed by flow cytometry. Cytotoxicity of CIK cells against EC9706 cells was measured using a standard LDH releasing assay. The effector cells were added to the target cells at E：T ratios of 10：1, 20： 1, 30：1, 40：1 and 50：1. In blocking experiments, NKG2D monoclonal antibody was added to CIK cells for 15 min before plat- ing at an E：T ratio of 30：1. Results： It was found that MIC and ULBP2 were expressed; however MICB, ULBP1 and ULBP3 were not detectable by EC9706 cells. At days 0 and 14, the percentage of CD3＋CD56＋ cells were （0.68±0.07）% and （56.55± 2.01）%, respectively （P〈0.05）, while the expression rates of NKG2D were （26.30± 1.12）% and （67.13± 1.34）%, respectively （P〈0.05）. The cytotoxicity of CIK cells against EC9706 cells were （28.81 ±0.47）% , （37.78±0.22）% , （44.31 ±1.06）% , （47.25±0.47）% and （57.62±0.94）%, respectively, at E：T ratios of 10：1, 20：1, 30：1, 40：1 and 50：1 （P〈0.05）. When the NKG2D molecules on bulk CIK cells were blocked by the NKG2D monoclonal antibody, the cytolytic activity of the CIK cell was significantly inhibited. Conclusion： During CIK cell expansion, NKG2D and CD3＋ CD56＋ are upregulated. NKG2D molecules play a role for cytotoxicity against EC9706 cells.