γδT细胞经唑来膦酸刺激后对骨肉瘤细胞的杀伤作用  

γδT cells stimulated by zoledronate kill osteosarcoma cells

作　　者：蒋晖[1] 徐强[1] 杨超[1] 曹贞国[2] 李朝旭[1] 叶招明[1] 

机构地区：[1]浙江大学附属第二医院骨科,浙江杭州310009 [2]徐州医学院附属第二医院骨科,江苏徐州221006

出　　处：《细胞与分子免疫学杂志》2010年第12期1195-1197,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30973444)

摘　　要：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。AIM： To investigate the cytotoxicity of human γδT cells from PBMCs stimulated by zoledronate against osteosarcoma cell line HOS in vitro and in vivo and evaluate the relavent pathways.METHODS： The peripheral blood mononuclear cells（PBMCs）of healthy donors were stimulated by single dose zoledronate and cultured in the present of IL-2 for two weeks,analysising the percentage of γδT cells on a FACSCalibur cytometer.Study the cytotoxicity of γδT cells against the osteosarcoma line HOS using LDH release assay kit.Pre-treatment of γδT cells with anti-human γδTCR antibody,anti-human NKG2D antibody and concanamycin A to bolck the relavent pathways for evaluating the mechenisms of its cytotoxicity.In vivo,BALB/c mice were inoculated subcutaneously osteosarcoma cell HOS for developing hypodermal tumors.And they were randomized into two groups： unteated group,γδT cell therapy group.Tumor volume and weight of the two groups were compared.RESULTS： After two weeks of culture,γδT cells from zoledronate-stimulated PBMCs could reach（95±3）%.When the E∶T as 6∶1,12∶1,25∶1,50∶1,the percentage of osteosarcoma cell HOS killed by γδT cells was 26.8%,31.5%,37.8%,40.9%,respectively.When anti-huma γδTCR antibody,anti-human NKG2D antibody and concanamycin A blocked the relavent pathways,the percentage was 32.3%,4.7%,16.7%（E∶T as 25∶1）,respectively.In vivo,the tumor inhibition rate of the group of γδT cell therapy was 42.78%.CONCLUSION： γδT cells derived from PBMCs stimulated by zoledronate can acquired pure γδT cells.And they show strong cytoxicity against osteosarcoma cell line HOS in vitro and in vivo.

关 键 词：ΓΔT细胞 唑来膦酸 细胞毒活性 裸鼠 免疫治疗 

分 类 号：R392.12[医药卫生—免疫学]