一种gamma-delta T细胞体外扩增体系的研制

Development of a Gamma-Delta T Cell Amplification System in Vitro

目的建立人单个核细胞培养的体外扩增gamma-delta T细胞体系,为肿瘤免疫细胞治疗奠定基础。方法采集体检健康人外周血,分离出单个核细胞,接种于含IL-2、唑来膦酸、 IL-18以及牻牛儿基牻牛儿基焦磷酸(GGPP)的体系中。根据加入激活剂种类的不同组合分为五个组,包括A组:唑来膦酸+IL-2; B组:唑来膦酸+IL-2+IL-18; C组:唑来膦酸+IL-2+GGPP; D组:唑来膦酸+IL-2+IL-18+GGPP; E组:不添加任何细胞因子作为激活剂。培养1周后,流式细胞术检测各组gamma-delta T细胞的纯度、颗粒酶B、穿孔素及CD56的阳性比例。以K562细胞为靶细胞, MTT实验观察细胞的体外杀伤活性。结果经过各组合细胞因子诱导7 d后, gamma-delta T细胞纯度均大于70%,细胞因子刺激组各组间gamma-delta T细胞纯度以及扩增倍数无显著差异(P>0.05)。经细胞因子刺激组的gamma-delta T细胞的颗粒酶B、细胞穿孔素以及CD56表达阳性率均显著高于E组,差异具有统计学意义(P <0.05)。经体外细胞因子处理后, gamma-delta T细胞不仅增殖数目与速度大大增加,而且一些具有杀伤作用的物质(颗粒酶B、细胞穿孔素与CD56)也大大增加,说明在细胞因子的参与下体外扩增对于肿瘤细胞的杀伤活性更强,杀伤效果更好。结论唑来膦酸、 IL-2、 IL-18、 GGPP可作为gamma-delta T细胞体外扩增体系中的重要组成部分,能够显著增强gamma-delta T细胞的毒活性,有效增强肿瘤细胞的杀伤活性。

Objective To establish a human mononuclear cell culture system to amplify gamma-delta T cells in vitro and to lay a foundation for tumor immunotherapy. Methods Peripheral blood samples from healthy individuals were collected and mononuclear cells were isolated. Inoculated with IL-2, Zoledronate acid, IL-18 and geranylgeranyl pyrophosphate （GGPP）, they were divided into five groups according to different combinations of activators. Group A： Zoledronic acid ＋ IL-2; group B： Zoledronic acid ＋ IL-2 ＋ IL-18; group C： Zoledronic acid ＋ IL-2 ＋ GGPP; group D： Zoledronic acid ＋ IL-2 ＋ IL-18 ＋ GGPP; group E： no cytokine was added as an activator. The purity, granzyme B, perforin and positive proportion of CD56 of gamma-delta T cells in each group was detected by flow cytometry after l week＇s culture. K562 cells were used as target cells to observe the cytotoxicity of K562 cells in vitro. Results After 7 days of induction by various combination of cytokines, the purity ofCD56 T cells was higher than 70%, and there was no significant difference in the purity and amplification of gamma-delta T cells among the cytokine stimulation groups （P〉0.05）. The granzyme B, perforin and positive proportion of CD56 of gamma-delta T cells in stimulation groups were significantly higher than those in group E, and the differences were statistically significant （P〈0.05）. After in vitro cytokine treatment, T cell proliferation and the number of gamma-delta increased greatly, and some with the killing effects of substances （granzyme B, perforin and CD56 cells） also increased significantly, indicated that the in vitro amplification with the participation of cytokines had stronger killing activity against tumor cells and better killing effect. Conclusions Zoledronic acid, IL-2, IL-18 and GGPP can be used as important components of gamma-delta T cell expansion system in vitro, which can significantly enhance the toxic activity of gamma-delta T cells, and effectively enhance the killing activity of tumor cells.