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目的:探讨外周血γδT细胞经唑来膦酸刺激后对骨肉瘤细胞株HOS的体内外杀伤作用及相关机制。方法:取健康人外周血5 mL,经唑来膦酸刺激后,在含IL-2的培养液中,培养14 d。用流式细胞仪测定γδT细胞所占的比例,用乳酸脱氢酶(LDH)释放法检测其对骨肉瘤细胞株HOS的体外杀伤作用,并用抗人γδTCR抗体、抗人NKG2D抗体和穿孔素/颗粒酶阻滞剂CMA分别预处理γδT细胞,观察杀伤作用的变化。另用骨肉瘤细胞株HOS注入BALB/c裸鼠皮下建立骨肉瘤裸鼠种植瘤模型。随机将裸鼠分为2组,分别为未治疗组、γδT细胞治疗组。观察裸鼠瘤体大小及称量瘤重。结果:PBMCs经唑来膦酸刺激14 d后γδT细胞阳性率达到(95±3)%。当效靶细胞比为6∶1、12∶1、25∶1、50∶1时,对HOS细胞的杀伤率分别为26.8%、31.5%、37.8%、40.9%。用Anti-γδTCR抗体、Anti-NKG2D抗体及CMA预处理γδT细胞后,当效靶细胞比为25∶1时,对HOS的杀伤率分别为32.3%、4.7%、16.7%。体内抑瘤实验中,γδT细胞治疗组的肿瘤生长抑制率为42.78%。结论:PBMCs经唑来膦酸刺激后,可获得高纯度的γδT细胞,在体内外其对骨肉瘤细胞株HOS有较强的杀伤作用。
OBJECTIVE: To investigate the cytotoxicity of γδT cells in vitro and in vivo on osteosarcoma cell line HOS stimulated by zoledronic acid. Methods: Peripheral blood of 5 mL healthy volunteers was stimulated with zoledronic acid and cultured in IL-2-containing medium for 14 days. The percentage of γδT cells was determined by flow cytometry and the killing effect on osteosarcoma cell line HOS was tested by lactate dehydrogenase (LDH) release assay. Anti-human γδTCR antibody, anti-human NKG2D antibody and perforin / Granzyme inhibitor CMA pretreatment γδT cells were observed changes in the killing effect. Another osteosarcoma cell line HOS injected BALB / c nude mice subcutaneously established osteosarcoma xenografts model in nude mice. Nude mice were randomly divided into two groups, untreated group, γδT cell treatment group. Observe the tumor size and weigh the tumor in nude mice. Results: The positive rate of γδT cells in PBMCs treated with zoledronic acid for 14 days was (95 ± 3)%. The killing rates of HOS cells were 26.8%, 31.5%, 37.8% and 40.9% when the effective target cell ratio was 6:1, 12:1, 25:1 and 50:1 respectively. Anti-γδTCR antibody, Anti-NKG2D antibody and CMA pretreatment γδ T cells, the effective target cell ratio of 25: 1, the HOS killing rates were 32.3%, 4.7%, 16.7%. In vivo antitumor experiments, the tumor growth inhibition rate of γδT cell treatment group was 42.78%. CONCLUSION: PBMCs stimulated by zoledronic acid can be obtained high purity γδT cells in vitro and in vivo osteosarcoma cell line HOS have a stronger killing effect.