Objective: To establish the culture technique for culturing γδ T cells in vitro and evaluate the basic characteristics, security and anti-tumor effect of the cultured γδ T cells. Methods: Phytohemagglutinin, zoledronic acid, interleukin-2 and interleukin -7 were used to induce the abundant expansion of peripheral blood mononuclear cells in vitro. Flow cytometry assay, in vitro killing assay and mouse model of human lung cancer were also adopted to assess the characteristics and the anti-tumor effect of cultured γδ T cells. Additionally, the contamination of exogenous agents and the acute toxicity of γδ T cells were determined. Results: After culturing 14-16 days in vitro, the total number of γδ T cells was more than 1.0×10(10). Among these γδ T cells, CD3(+) γδ TCR(+) cells accounted for more than 90%. None of contaminations of bacteria, fungi, mycoplasma and virus were observed. At effect target ratio (E/T ratio) of 50/1, killing efficiency of γδ T cells cultured in vitro to SK-MES-1, Ho8910, A549 and K562 reached more than 65%. In vivo experiments showed that the tumor volume of γδ T-treated mice was (828.99±61.05) mm(3,) significantly lower than (1 723.51±84.30) mm(3) of the control mice (P<0.05). Meanwhile, no acute toxicity effect was observed in γδ T cells treated mice. Conclusion: The number, purity and activity of γδT cells cultured in our institute can reach the requirement of clinical application, and the γδT cells also display strong cytotoxic activity against tumor cells such as lung cancer, ovarian cancer and leukemia.
目的： 探讨建立体外培养γδ T细胞的方法及其体内外抗肿瘤活性及安全性。 方法： 应用植物血凝素、唑来膦酸、白细胞介素2(IL－2)和IL－7等刺激外周血单个核细胞，在体外进行大量扩增。采用流式细胞术、体外杀伤实验和荷人肺癌小鼠模型检测γδ T细胞的特点和抗肿瘤活性，采用病毒检测试剂盒检测γδ T细胞中外源性因子的污染情况。 结果： 体外培养14～16 d后，γδ T细胞扩增总数>1.0×10(10)个，其中CD3(＋) γδ TCR(＋)细胞所占比例>90%，无细菌、真菌、支原体和病毒因子等污染。体外扩增后的γδ T细胞对肺鳞癌细胞SK－MES－1、卵巢癌细胞Ho8910、肺腺癌细胞A549和慢性髓原白血病细胞K562均具有较强的细胞毒性，杀伤效率均>65% (效靶比为50∶1)。实验组和对照组小鼠的肿瘤体积分别为(828.99±61.05)mm(3)和(1 723.51±84.30)mm(3)，差异有统计学意义(P<0.05)。输注γδ T细胞的小鼠无急性不良反应。 结论： 培养的γδ T细胞具有足够的数量、高纯度、较强的活性、良好的体内外抗肿瘤活性及安全性，对肺癌、卵巢癌和白血病肿瘤细胞具有杀伤活性。.
Keywords: Anti-tumor effect; Lung neoplasms; Tumor immunotherapy; γδ T cells.