目的：探究IL-15、4-1BBL基因修饰的人白血病K562细胞（modi-K562细胞）联合IL-2体外高效扩增肾细胞癌患者自体自然杀伤（natural killer，NK）细胞的方法，研究扩增前后NK细胞对肾癌细胞株786-O的杀伤作用。方法：10例肾癌患者的外周血单个核细胞（peripheral blood mononuclear cell，PBMC）与modi-K562细胞在含不同浓度IL-2培养液中共育14 d，采用流式细胞术、Calcein-AM释放实验检测NK细胞的扩增情况、免疫表型及对肾癌786-O细胞的杀伤作用。结果：modi-K562细胞联合IL-2可有效扩增NK细胞，300 U/ml IL-2培养14 d时，NK细胞扩增（202.4±12.8)倍。在效靶比为20∶1时，扩增后NK细胞对786-O细胞的杀伤率为（72.0±4.3）%，显著高于扩增前NK细胞的杀伤率（34.2±3.6）%（P<0.01）。结论：IL-15、4-1BBL〖基因修饰的K562细胞联合IL-2在体外能有效扩增肾细胞癌患者NK细胞，扩增后NK细胞对肾癌786-O细胞的杀伤作用显著增强。

Objective: To explore the expansion method of autologous NK cells from renal cell carcinoma (RCC) patients based on IL-15- and 4-1BBL-modified K562 cells (modi-K562 cells) combined with IL-2 and to investigate their cytotoxicity against RCC 786-O cell lines. Methods: Peripheral blood mononuclear cells (PBMCs) from 10 RCC patients were co-cultured with modi-K562 cells in medium containing various concentrations of IL-2 for 14 d. The expansion, immune phenotype and cytotoxicity against 786-O cells were analyzed by flow cytometry and Calcein-AM release assay. Results: modi-K562 combined with IL-2 effectively amplified NK cells in vitro. The number of NK cells from 300 IU/mL IL-2 co-culture system for 14 d had amplified on an average of （202.4±12.8) fold. Moreover, the expanded NK cells significantly increased cytotoxicity against 786-O cells. At an effect/target (E∶T) ratio of 20∶1, (72.0±4.3)% of the targets were killed by the expanded NK cells, whereas unexpanded ones killed only (34.2±3.6)% of targets (P<0.01). Conclusion: modi-K562 cells combined with IL-2 can effectively stimulate the expansion of RCC patients’ NK cells in vitro, which significantly augment the cytotoxicity against RCC 786-O cells.

国家自然科学基金资助（No. 81071856，No.30973450）；上海市浦江人才计划资助（No. 11PJ1407900）；上海市第十人民医院基金资助（No.10RD103，No.11SC103）