γδT细胞体外扩增方法的建立及其抗肿瘤活性  

Establishment of the culture system of γδ T cells in vitro and the anti-tumor effect

作　　者：谢蕾 陈稳[2] 王亮[2] 程民[2] 胡世莲[2] 沈干[1] Xie Lei;Chen Wen;Wang Liang;Cheng Min;Hu Shilian;Shen Can(Department of Geriatric Medicine, Anhui Province Hospital Affiliated Anhui Medical University, Hefei 230001, China;Anhui Provincial Key Laboratory of Tumor Immunotherapy and Nutrition Therapy, Anhui Province Hospital Affiliated Anhui Medical University, Hefei 230001, China)

机构地区：[1]安徽医科大学附属安徽省立医院老年医学科,合肥230001 [2]安徽医科大学附属安徽省立医院肿瘤免疫与营养治疗安徽省重点实验室,合肥230001

出　　处：《中华肿瘤杂志》2018年第4期247-251,共5页Chinese Journal of Oncology

基　　金：国家自然科学基金（81471552）;安徽省重点实验室绩效项目（1606c08236）;安徽省科技攻关计划（1501041142）

摘　　要：目的探讨建立体外培养18T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18T细胞的特点和抗肿瘤活性，采用病毒检测试剂盒检测^y6T细胞中外源性因子的污染情况。结果体外培养14-16d后，16T细胞扩增总数〉1．0×10^10个，其中CD3＋γδTCR＋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18T细胞对肺鳞癌细胞SK-MES．1、卵巢癌细胞H08910、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（1723．51±84．30）mm^3，差异有统计学意义（P〈0．05）。输注18T细胞的小鼠无急性不良反应。结论培养的γδT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。Objective To establish the culture technique for culturing γδT cells in vitro and evaluate the basic characteristics, security and anti-tanlor effect of the cultured γδT 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 γδT 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 γδT ceils 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 γδT ceils 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） mm3, significantly lower than （1 723.51±84.30） mm3 of the control mice （P〈0.05）. Meanwhile, no acute toxicity effect was observed in γδT 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.

关 键 词：肺肿瘤 肿瘤免疫治疗 ΓΔT细胞 抗瘤活性 

分 类 号：R730[医药卫生—肿瘤]