目的：建立γδT细胞培养体系，探讨γδT细胞对不同血液肿瘤细胞的杀伤活性。方法：选取2016年1月至2016年4月在解放军第307医院造血干细胞移植科收治的4例B细胞淋巴瘤患者和该院5例体检健康志愿者，分别分离其外周血单个核细胞(peripheral blood mononuclear cell, PBMC)，用唑来磷酸（zoledronate, Zol）和IL-2体外扩增γδT细胞，采用流式细胞术检测γδT细胞对血液肿瘤细胞Jurkat、THP-1、HL-60、K562、Raji、U-937和RPMI-8226的杀伤活性，比较CIK、NK和γδ T三种细胞对K562细胞的杀伤作用，检测γδT细胞IFN-γ、TNF-α的分泌水平及CD107a分子的表达水平。结果:成功在体外扩增外周血γδT细胞；γδT细胞对Jurkat、THP-1、HL-60、K562、U-937和RPMI-8226细胞均有明显的杀伤活性(P<005)；γδT细胞对K562细胞的杀伤活性与NK细胞无统计学差异，但明显强于CIK细胞(P<0.01)；随着与K562细胞共孵育时间的延长，γδT细胞分泌IFN-γ的水平呈现出时间依赖性增加，TNF-α在8 h后逐渐增加；与K562细胞或HL-60细胞共孵育后，γδT细胞CD107a分子的表达水平均显著增加(P<0.01)。结论: 体外扩增的γδT细胞对血液肿瘤细胞具有较高的杀伤活性，为血液肿瘤的细胞免疫治疗提供实验依据。

Objective:To establish culture system of γδT cell and to explore killing activity of the γδT cells against different human hematologic neoplasms cells. Methods:Four patients with lymphoma who were hospitalized in the Department of Hematopoietic Stem Cell Transplantation, the 307th Hospital of PLA and five healthy volunteers for physical examination during January to April 2016 were selected and their peripheral blood mononuclear cell (PBMC) were isolated respectively. γδT cells were amplified in vitro with zoledronate (Zol) and IL-2, and killing activities of the γδT cells against hematologic neoplasms Jurkat, THP-1, HL-60, K562, Raji, U-937 and RPMI-8226 line cells were investigated by Flow cytometry assay. Killing effects of CIK cells, NK cells and the γδT cells on K562 cells were compared; and the expression levels of IFN-γ, TNF-α and secretion level of CD107a molecule in the γδT cells were detected. Results: The γδT cells were successfully amplified from peripheral blood in vitro. The γδT cells showed obvious killing activities against all of the Jurkat, THP-1, HL-60, K562, U-937 and RPMI-8226 cells (P<0.05). There was not a statistical difference between killing activities of the γδT and NK cells against the K562 cells (P>0.05), but killing activity of the γδT cells against K562 cells was higher than that of CIK cells (P<0.01). With extending the co-incubation time with K562 cells, level of IFN-γ secreted by the γδT cells increased as a time-dependent manner, in addition, level of TNF-α in the γδT cells gradually increased after 8 h of the co-incubation. After co-incubation of the γδT cells with the K562 or the HL-60 cells, expression level of CD107a molecule in the γδT cells was significantly up-regulated (P<0.01). Conclusion：The γδT cells amplified showed higher killing activity against hematologic neoplasms cells, which could provide experimental evidence for cellular immunotherapy of hematologic neoplasms.

国家高技术研究发展计划（863计划）资助项目（2013AA0200103）；北京市科委首都特色课题基金资助项目（Z161100000516184）