目的：建立体外扩增人脐带血来源的NK细胞的方法，并研究其对多株人食管癌细胞系和食管癌患者原代肿瘤细胞的杀伤作用。方法：分离人脐带血单个核细胞，加入放射线灭活的人白血病K562细胞及IL-2、IL-15、IL-18细胞因子体外扩增培养2周，诱导NK细胞。采用流式细胞术检测NK细胞的扩增效果及其产生的IFN-γ等细胞因子杀伤靶细胞K562的能力，并检测体外扩增的人脐带血来源NK细胞对多种食管癌细胞系和食管癌患者原代肿瘤细胞凋亡的影响。结果：利用放射线灭活的K562细胞联合多种细胞因子，可以有效的扩增人脐带血来源的NK细胞。体外培养2周后NK细胞比率可达80%左右，并对靶细胞K562有明显促凋亡作用。此外，体外扩增的人脐带血来源NK细胞能够促进6株人食管癌细胞系和食管癌患者原代肿瘤细胞的凋亡。结论：本研究建立了体外扩增人脐带血NK细胞的方法，并证实这些NK细胞对食管癌细胞的促凋亡作用，可能为食管癌的免疫治疗提供新的手段。

Objective:To establish a method for amplification ex vivo of natural killer (NK) cells derived from human umbilical cord blood and to investigate cytokilling effect of the NK cell on multiple cell lines of human esophageal cancer and primary generation tumor cells of the patients with esophageal cancer. Methods: Human cord blood mononuclear cells (CBMC) were isolated and, irradiated inactivation human leukemia K562 cell and IL-2, IL-15, IL-18 cytokines were added into to amplify NK cells in culture ex vivo for 2 weeks. Amplification results of the NK cells and killing ability of secreted cytolines IFN-γ and so on against the target K562 cell were detected by Flow cytometry assay. Effect of NK cell amplificated ex vivo from human umbilical cord blood on apoptosis of various esophageal cancer cell lines and primary generation cancer cell of the patients with esophageal cancer were examined. Results: Irradiated inactivation K562 cell combined with multiple cytokines can effectively amplified NK cell derived from human umbilical cord blood. After culture ex vivo for 2 weeks, percentage of the NK cell can reach about 80% and had obviously promoting apoptosis of the K562 cell. In addition, amplification ex vivo NK cell derived from human umbilical cord blood can promote apoptosis of the 6 cell lines of human esophageal cancer and the primary generation cancer cell of the patients with esophageal cancer. Conclusion: In the study, the method of amplification ex vivo of NK cells derived from human umbilical cord blood was successfully established. The promoting apoptosis effect of the NK cells on esophageal cancer cells might be confirmed. It could provide a novel approach for immunotherapy of esophageal cancer.

河南省基础与前沿技术研究计划基金资助项目（No.142300410387.0）