γδT细胞的体外扩增及对不同肿瘤细胞的杀伤性研究

doi:10.3760/cma.j.issn.1673422X.2018.08.001

摘要/Abstract

摘要： 目的通过体外制备γδT细胞，研究γδT细胞对不同肿瘤细胞的杀伤性。方法采集2018年1月至3月第三军医大学新桥医院招募的20例健康志愿者外周静脉血，使用唑来膦酸联合白细胞介素2（IL2）对人外周血单个核细胞（PBMC）进行诱导扩增，获取γδT细胞。流式细胞仪检测第0、7、10、14、16天的细胞纯度。使用CCK8法检测其对肿瘤细胞的杀伤性，以培养第14天的细胞作为效应细胞，以乳腺癌BCap37、肝癌Bel7402肿瘤细胞作为靶细胞，分别在效靶比5∶1、10∶1、20∶1下进行细胞杀伤活性检测。结果流式细胞检测第0、7、10、14、16天γδT细胞的纯度分别为（3.35±1.32）%、（50.76±5.76）%、（80.43±4.53）%、（90.56±3.34）%、（89.54±4.42）%，差异有统计学意义（F=18.431，P=0.012）。5∶1组、10∶1组、20∶1组的γδT细胞对BCap37的杀伤率分别为（31.3±2.0）%、（48.7±1.6）%、（71.3±2.4）%，差异有统计学意义（F=16.724，P=0.016），进一步两两比较，10∶1组高于5∶1组（P=0.013），20∶1组高于5∶1组（P=0.017），20∶1组高于10∶1组（P=0.011），杀伤率随着效靶比的增高而增大。5∶1组、10∶1组、20∶1组的γδT细胞对Bel7402的杀伤率分别为（34.5±2.0）%、（52.4±1.9）%、（74.5±1.6）%，差异有统计学意义（F=18.253，P=0.013），进一步两两比较，10∶1组高于5∶1组（P=0.015），20∶1组高于5∶1组（P=0.012），20∶1组高于10∶1组（P=0.015），杀伤率随着效靶比的增高而增大。结论使用唑来膦酸联合IL2诱导PBMC可以获得高纯度的γδT细胞，扩增的γδT细胞对BCap37和Bel7402肿瘤细胞有明显的杀伤作用。

关键词: γδT细胞, 唑来膦酸, 杀伤功能

Abstract: ObjectiveTo study the killing effect of γδT cells on different tumor cells by inducing and expanding γδT cells in vitro. MethodsCollected the peripheral venous blood of 20 healthy volunteers recruited at Xinqiao Hospital of Third Military Medical University from January to March 2018. Peripheral blood mononuclear cells (PBMCs) were induced and expanded using zoledronic acid combined with interleukin2 (IL2) to obtain γδT cells. The cell purity was detected on day 0, 7, 10, 14 and 16 using flow cytometry. The killing activity was detected by CCK8 kit. Cells cultured on the 14th day were used as effector cells, and breast cancer cell BCap37 and liver cancer cell Bel7402 were used as target cells, and the cell killing activity was detected at the effective target ratio (E∶T) of 5∶1, 10∶1 and 20∶1 respectively. ResultsThe purity of γδT cells were respectively (3.35±1.32)%, (50.76±5.76)%, (80.43±4.53)%, (90.56±3.34)%, (89.54±4.42)% on day 0, 7, 10, 14 , 16, with significant difference (F=18.431, P=0.012). The efficiency of γδT cells killing BCap37 tumor cells were (31.3±2.0)% (E∶T=5∶1), (48.7±1.6)% (E∶T=10∶1), (71.3±2.4)% (E∶T=20∶1) respectively, with significant difference (F=16.724, P=0.016), further comparison between each two groups: 10∶1 vs. 5∶1 (P=0.013), 20∶1 vs. 5∶1 (P=0.017), 20∶1 vs. 10∶1 (P=0.011). The killing rate increased with the increase of target ratio. The efficiency of γδT cells killing Bel7402 tumor cells were (34.5±2.0)% (E∶T=5∶1), (52.4±1.9)% (E∶T=10∶1), (74.5±1.6)% (E∶T=20∶1) respectively, with significant difference (F=18.253, P=0.013), further comparison between each two groups: 10∶1 vs. 5∶1 (P=0.015), 20∶1 vs. 5∶1 (P=0.012), 20∶1 vs. 10∶1 (P=0.015). The killing rate increased with the increase of target ratio. ConclusionWe can obtain high purity γδT cells using zoledronic acid combined with IL2 induced PBMCs, and the amplified γδT cells have significant killing effect on BCap37 and Bel7402 tumor cells.

Key words: γδT cells, Zoledronic acid, Killing function

张光辉, 邵小燕, 严小敏, 魏士钧. γδT细胞的体外扩增及对不同肿瘤细胞的杀伤性研究[J]. 国际肿瘤学杂志, 2018, 45(8): 449-452.

ZHANG Guang-Hui, SHAO Xiao-Yan, YAN Xiao-Min, WEI Shi-Jun. In vitro expansion of γδT cells and their killing effect on different tumor cells[J]. Journal of International Oncology, 2018, 45(8): 449-452.

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