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目的探讨唑来膦酸对骨肉瘤患者外周血单个核细胞(PBMCs)来源γδT细胞杀伤骨肉瘤作用的影响。方法使用唑来膦酸联合IL-2体外扩增原发性、复发性和转移性骨肉瘤患者PBMCs来源γδT细胞,LDH法检测γδT细胞的杀伤活性,ELISA法检测γδT细胞分泌IFN-γ情况,并应用不同的抗体确定γδT细胞识别、杀伤骨肉瘤细胞的途径。建立裸鼠HOS骨肉瘤移植瘤模型,尾静脉分别注射生理盐水、唑来膦酸、γδT细胞及唑来膦酸联合γδT细胞,观察并比较不同方法对裸鼠肿瘤生长的抑制作用。结果骨肉瘤患者PBMCs体外经过唑来膦酸联合IL-2刺激可高度选择性扩增γδT细胞,并且扩增后的γδT细胞具有杀伤骨肉瘤细胞的能力。唑来膦酸预处理骨肉瘤细胞后可显著增强γδT细胞的杀伤活性,γδT细胞识别、杀伤唑来膦酸预处理骨肉瘤细胞主要通过TCR途径和穿孔素-纤溶酶途径,NKG2D途径和TRAIL途径发挥作用较小。体内实验表明,唑来膦酸联合γδT细胞治疗对肿瘤生长的抑制作用强烈而持久,显著优于单独唑来膦酸治疗组和单独γδT细胞治疗组。结论唑来膦酸能够促进骨肉瘤患者PBMCs来源γδT细胞的增殖,并能够增强γδT细胞的抗骨肉瘤作用。
Objective To investigate the effect of zoledronic acid on the cytotoxicity of γδT cells derived from peripheral blood mononuclear cells (PBMCs) from patients with osteosarcoma. Methods PBMCs derived from patients with primary, recurrent and metastatic osteosarcoma were amplified by zoledronic acid combined with IL-2. The cytotoxicity of γδT cells was detected by LDH and the secretion of IFN-γ by γδT cells by ELISA. And different antibodies were used to determine the way of γδT cells to recognize and kill osteosarcoma cells. The nude mice model of HOS osteosarcoma xenografts were established. The tail vein was injected with normal saline, zoledronic acid, γδT cells and zoledronic acid with γδT cells. The inhibitory effects of different methods on tumor growth in nude mice were observed and compared. Results PBMCs from osteosarcoma were highly selective for γδT cells stimulated by zoledronic acid and IL-2, and the expanded γδT cells had the ability to kill osteosarcoma cells. Zoledronic acid pretreatment of osteosarcoma cells can significantly enhance the γδT cytotoxic activity, γδ T cells recognize and kill zoledronic acid pretreatment of osteosarcoma cells mainly by TCR and perforin-plasmin pathway, NKG2D pathway and TRAIL Way to play a less important role. In vivo experiments showed that the combination of zoledronic acid and γδT cells had a strong and lasting inhibitory effect on tumor growth, which was significantly better than that of zoledronic acid alone and γδT cells alone. Conclusion Zoledronic acid can promote the proliferation of γδT cells derived from PBMCs in osteosarcoma patients and enhance the anti-osteosarcoma effect of γδT cells.