树突状细胞对自体自然杀伤细胞体外扩增及功能的影响

Effect of Dendritic Cells on Expansion and Function of Autologous Natural Killer CellsIn Vitro

本研究探讨树突状细胞(DC)对自体自然杀伤(NK)细胞体外扩增和功能的影响及其机制。用干细胞培养液(SCGM)在37℃、5%CO2、饱和湿度培养条件下以rhIL-2体外扩增NK细胞10天后,将自体DC与NK细胞以5∶1(5∶1组)和1∶1(1∶1组)的比例混合继续培养。14、21天时计算5∶1组、1∶1组和对照组的NK细胞扩增倍数,流式细胞术测定细胞表面CD3、CD56/16的表达,MTT法检测NK细胞的功能,ELISA法检测各培养组上清液中TNF-α和IL-12p70的含量。结果表明:14天时5∶1、1∶1组和对照组的扩增倍数分别为29.25±4.01、21.23±2.91和16.26±1.58,3组间比较差别有统计学意义(p<0.05)。同组不同时间比较,14天时扩增倍数最高(p<0.05)。14天时3组CD3-、CD56/16+表达率分别为(64.6±7.8)%、(50.6±8.7)%和(34.8±5.1)%,5∶1组表达率最高(p<0.05)。14天时3组对K562细胞的杀伤率分别为(87.4±6.8)%、(75.4±6.3)%和(63.7±3.8)%,5∶1组杀伤活性明显高于其它2组(p<0.05)。5∶1组上清液中TNF-α和IL-12p70的含量均分别高于其它各组(p均<0.05)。结论:DC与NK细胞混合培养,能以比例依赖的方式增加NK细胞的扩增倍数,增强NK细胞的功能。DC增加NK细胞的扩增倍数与DC分泌的IL-12有关,NK细胞功能增强与NK细胞分泌的TNF-α增高有关。

The objective of this study was to investigate the effect of dendritic cells （DCs） on expansion and function of autologous natural killer （NK） cells and its mechanism in vitro. NK cells were expanded from peripheral blood mononuclear cells （PBMNCs） of healthy volunteers in stem cell growth medium （SCGM） supplemented with rhIL-2 （control group）in 24-well culture plates at 37℃ in a humidified CO2-containing atmosphere. NK cells were cultured with autologous DCs in the ratio of 5 to 1 （group 5：1 ）or 1 to 1 （group 1：1 ）from day 10 after expansion. Total cells of every group were counted and the expression of CD3 ,CD16/56 on the surface of NK cells was assayed by flow cytometry on days 7,14 and 21 to calculate the expansion of NK cells . Cytotoxicity of expanded NK cells against K562 cells was assayed by MTT method. TNF-α and IL-12p70 were detected in culture supernatants by sandwich ELISA. The results indicated that the expansion and cytotoxicity of NK cells were improved after mixed with autolgous DCs. Further- more, when DCs were mixed with NK cells, the ratio of DCs to NK cells was higher, the expansion and cytotoxicity NK cells were higher. On day 14, the expansion multiple in control, group 5：1 and group 1：1 were 16.26 ± 1.58,29.25 ± 4.01 and 21.23 ±2.91 respectively. The expansion multiple of group 5：1 was much higher than that of the other two groups （p 〈0.05）. The expressions of CD3^- ,CD56/16^＋ on surface of NK cells in control, group 5：1 ,group 1：1 were （34.8 ±5.1 ） %, （64.6 ± 7.8） % and （50.6 ± 8.7） % respectively and that of group 5：1 was the highest （p 〈0. 05）. The cytotoxicities against K562 cells in control, group 5： 1 and group 1 ： 1 were （ 63.7 ± 3.8 ） %, （ 87.4 ± 6.8 ） % and （75.4±6.3）% respectively. The cytotoxicity of group 5：1 was higher than that in the other two groups also （p 〈 0. 05）. TNF-α and IL-12p70 levels in culture supernatants when DCs and NK cells were mixed in the ratio of 5 to 1 were much higher than those in culture supernatants of DCs and NK cells alone or in culture supernatants when DCs and NK cells were mixed in the ratio of 1 to 1 （p 〈0.05）. It is concluded that the expansion and cytotoxicity of NK cells can be improved by DCs and it depended on the mixed ratio of DCs to NK cells. The elevated expansion of NK cells by DCs bears relation to IL-12 produced by DCs. The enhanced cytotoxicity of NK cells is associated with TNF-α secreted by NK cells.