辐照后的AlloMNCs诱导NK细胞体外扩增

Proliferation of human natural killer cells induced with irradiated allogeneic peripheral blood mononuclear cells ex vivo

目的探讨照射后的同种异体外周血单个核细胞(allogeneic PB-MNCs,AlloMNCs)对人自然杀伤细胞(NK)体外扩增的影响,为NK细胞的过继免疫治疗奠定基础。方法 Ficoll密度梯度离心法分离健康成人外周血单个核细胞(PBMCs),30、50和70 Gyγ射线照射后的AlloMNCs作为饲养细胞,分别加入上述PBMCs,在添加细胞因子的X-VIVO15TM无血清培养基中进行培养扩增,同时选择未添加饲养细胞的作为对照组;台盼蓝拒染法进行活细胞计数;流式细胞术检测CD56+CD3-NK细胞纯度;MTT法检测细胞杀伤活性。结果加入饲养细胞的PBMCs在添加了细胞因子的X-VIVO15TM无血清培养基中可培养21 d,30 Gy照射后的AlloMNCs作为饲养细胞时细胞扩增最明显,在第21天时扩增了270.3±10.4倍,CD56+CD3-NK细胞纯度由培养前的(9.7±2.4)%提高到扩增后的(56.5±7.8)%,与无饲养细胞的对照组相比较,差异均有统计学意义(P<0.05)。细胞杀伤实验表明,当效靶比为10∶1时,添加30Gy照射后的AlloMNCs刺激扩增的NK细胞对K562和HO8910的杀伤率平均达到59.7%、63.2%,分别与对照组的28.3%、32.2%相比较,差异均有统计学意义(P<0.05)。结论 30 Gy照射后的AlloMNCs可作为饲养细胞在体外促进NK细胞的增殖,为NK细胞的肿瘤过继免疫治疗提供了一种简单有效的扩增NK细胞的方法。

The aim of this study is to explore effect of irradiated allogeneic peripheral blood mononuclear cells（AlloMNCs） on the proliferation of human natural killer cells.We isolated peripheral blood mononuclear cells（PBMCs） by Ficoll density-gradient centrifugation,which then feed with AlloMNCs irradiated with different doses of gamma ray.After that,the PBMCs and the feeder cells were cocultivated in X-VIVO15TM medium supplemented with cytokines,with PBMCs cultured without feeder cells as control.We counted the cells by using trypan blue exclusion,detected the NK cell purity by FCM,and measured the cytotoxicity of the expanded cells by MTT method.During the whole expansion period of approximately 21 days,the number of PBMCs with 30 Gy irradiated feeder cells increased observably and expanded 270.3 ±10.4 fold,containing（56.5±7.8） % CD56＋CD3-NK cells,which was significantly higher than that in control group（9.7% ± 2.4%）（P〈0.05）.The expanded cells population with irradiated（30 Gy） feeder cells displayed high cytotoxicity against K562 and HO8910 with a mean specific lysis of 59.7% and 63.2% at 10∶1 effector to target ratio which were significantly higher those of control group（28.3% and 32.2%,respectively）（P〈0.05）.We conclude that NK cells could be expanded from PBMCs with 30 Gy irradiated AlloMNCs.This work provide a simple and efficient way of expanding human NK cells for the adoptive immunotherapy of tumor with NK cells.