不同刺激因子组合诱导NK细胞体外扩增及其功能的研究  

Expansion and function of human NK cells induced with different stimulating factor combinations ex vivo

作　　者：马杰[1] 赵卫东[1] 张丽娜[1] 周虎[1] 丁玉兰[1] 宣恒华[1] 桂云[1] 金夏[1] 

机构地区：[1]安徽医科大学附属省立医院妇产科,合肥230001

出　　处：《免疫学杂志》2011年第6期510-513,518,共5页Immunological Journal

基　　金：安徽省科技攻关课题(08010302101)

摘　　要：目的探讨不同刺激因子组合对人自然杀伤细胞(NK细胞)体外扩增及其功能的影响。方法 VarioMACS分选健康成人外周血单个核细胞(PBMC)中的NK细胞,依据添加刺激因子的不同分为A组(IL-2)、B组(IL-2+IL-15)、C组(IL-2+IL-15+饲养细胞),饲养细胞为30 Gyγ射线照射后的同种异体外周血单个核细胞(allogeneic PB-MNCs,AlloMNCs),未添加任何刺激因子的细胞的作为对照组,在干细胞生长培养基(SCGM)中进行培养扩增14 d,第1天添加PHA及OKT3,第5天离心洗去;台盼蓝拒染法进行活细胞计数;流式细胞术检测分选及扩增前后CD56+CD3-NK细胞纯度;改良的MTT法检测NK细胞对K562、HO8910及PBMC的杀伤活性。结果经VarioMACS免疫磁珠阴性分选后NK细胞纯度由分选前的(9.2±2.9)%提高到(93.5±3.2)%,培养14 d后,除对照组NK细胞纯度降低外,其余组与扩增前无明显差异(P>0.05)。细胞扩增倍数分别为17.2±1.7、51.3±6.6和82.4±9.8倍,均显著高于对照组(5.7±1.2)(P<0.01)。实验组组间比较,C组明显高于A组和B组(P<0.01)。细胞杀伤实验表明,当效靶比为10∶1时,各组的杀伤活性显示为C组>B组>A组>对照组,C组扩增的NK细胞对K562及HO8910的杀伤率可分别达到(70.1±8.9)%和(64.6±6.2)%,显著高于对照组、A组(P<0.01)和B组(P<0.05),对PBMC的杀伤率仅为(4.2±1.2)%。结论经VarioMACS分选获得的NK细胞,在体外使用SCGM培养基培养,添加IL-2、IL-15和照射后AlloMNCs作为刺激因子,可获得高效扩增和有效活化的NK细胞,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高纯度NK细胞的方法。This study was purposed to explore the effects of different stimulating factor combinations on the expansion and function of human natural killer cells.Highly purified NK cells（93.5% ± 3.2%） were isolated from peripheral blood mononuclear cells（PBMCs） by VarioMACS.According to the different stimulating factors,the ex-perimental groups were divided into group A（IL-2）,group B（IL-2 ＋ IL-15）,and group C（IL-2 ＋ IL-15 ＋ feed-er cells）.The feeder cells are allogeneic peripheral blood mononuclear cells（AlloMNCs） irradiated with 30 Gy of gamma ray.The group in which NK cells were cultured without any stimulating factors was used as control.The cells of all groups were cultivated for 14 days in stem cell growth medium（SCGM） with PHA and OKT3 at the first 5 days.Then the viable cells were counted by using trypan blue exclusion,the purity of NK cells were de-tected with FCM,and the cytotoxicity of NK cells was detected by using cell counting Kit-8.During the whole expansion period of 14 days,the purity of NK cells had no observably change except for the control.But the NK cells expanded 17.2±1.7 fold,51.3±6.6 fold,and 82.4±9.8 fold in the three experimental groups respectively,all higher than that in the control（5.7±1.2）（P0.01）.And group C was higher than group A or group B（P 0.01）.As for the cytotoxicity of expanded NK cells,group C group B group A at 10：1 of effector to target.The cells of group C displayed the highest cytotoxicity against K562 and HO8910 with specific lysis of（70.1±8.9）% and （64.6±6.2）%,as compared with the control group（P0.01）,group A（P0.01）and group B（P0.05）.Whilethe cytotoxicity against PBMC was（4.2±1.2）%only.It is concluded that the NK cells isolated from PBMCs byVarioMACS co-cultivated with IL-2,IL-15 and irradiated AlloMNCs in SCGM could obtain the high expansionand effective activation.This study provides a simple and efficient way of obtaining human NK cells for theadoptive immunotherapy of tumor.

关 键 词：自然杀伤细胞 扩增 饲养细胞 刺激因子组合 免疫治疗 

分 类 号：R392.33[医药卫生—免疫学]