γδT细胞对血液肿瘤细胞的体外杀伤活性

Killing activity in vitro of γδT cells against human hematologic neoplasms cells

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γδT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 307th 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-γsecreted 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.