流式细胞术三种染色方法检测体外纯化扩增的NK细胞的细胞毒作用比较  

Comparison of Ex Vivo Expanded and Highly Purified NK Cell-Mediated Cytotoxicity Detected by 3 Different Staining Methods of Flow Cytometry

作　　者：郭继强[1] 韩亚萍[1] 李芳[1] 赵晴[1] 贾原[1] 靳保利 张俊萍[1] 

机构地区：[1]山西医学科学院山西大医院生物治疗科,山西太原030032 [2]山西医科大学研究生院,山西太原030032

出　　处：《中国实验血液学杂志》2016年第6期1691-1697,共7页Journal of Experimental Hematology

基　　金：山西省生物治疗基础建设示范平台基金项目(2014091105-0101)

摘　　要：目的:比较流式细胞术3种不同染色方法检测体外诱导扩增后NK细胞的细胞毒活性效果。方法:收集健康志愿者外周血,体外诱导扩增NK细胞,在培养第17天后分为3组,每组按10∶1、20∶1、40∶1 3个效靶比混合,共同培养4 h,并分组用3种不同方法染色后经流式细胞术检测。A组:CFSE/Annectin-V/7-AAD三色荧光染色法;B组:Annectin-V/PI双色荧光染色法;C组:CFSE/PI双色荧光染色法。结果:培养17 d后,NK细胞(CD3-CD56+)由培养第0天(16.34±10.51)%增加到(83.63±10.63)%(P<0.05)。3种染色方法检测结果均显示,扩增后NK细胞对K562细胞具有显著细胞毒活性:A组NK细胞对K562细胞的细胞毒活性均明显高于B和C组(P<0.05),当效靶比为10∶1时,A、B和C 3个组细胞毒活性分别为(36.56±3.69)%、(22.35±2.71)%和(10.85±2.09)%;当效靶比为20∶1时细胞毒活性分别为(47.83±5.52)%、(39.07±5.55)%和(29.61±4.81)%;当效靶比为40∶1时细胞毒活性则分别为(67.7±4.77)%、(51.51±4.43)%和(44.12±5.62)%,差异均有显著统计学意义(P<0.05)。结论:健康者外周血经体外分离培养,可得到高纯度NK细胞;流式细胞检测技术CFSE/Annectin-V/7-AAD三色荧光染色法能特异性地和灵敏地检测NK细胞的细胞毒活性。Objective：To compare the cytotoxicity of ex vivo expanded NK cells detected by flow cytometry with 3different staining methods.Methods：NK cells were collected from peripheral blood on the 17-（th） day after culture.The cultured cells were divided into 3 groups：group A,B,and C.The cells in group A were stained with CFSE/AnnectinV/7-AAD;the cells in group B were stained with Annectin-V/PI,and the cells in group C cells were stained with CFSE/PI.The E：T ratios in 3 groups were 10：1,20：1 and 40：1,respectively,the K562 cells were incubated with NK cells for 4 hrs.Results：The purity of NK cells（CD3^- CD56^＋） reached to（16.34 ± 10.51）%on day 0 and to（83.63± 10.63）%on the day 17 after incubation（P〈0.05）;the cytotoxicity of group A was significantly higher than thay of group B at different E：T ratio（P〈0.05）.The cytotoxicities in A,B,C groups at E：T ratio = 10：1 were（36.56 ±3.69）%,（10.85 ±2.09）%and（22.35 ±2.71）%respectively;the cytotoxicities in A,B,C groups at E：T ratio =9：1 were（47.83 ±5.52）%,（39.07 ±5.55）%and（29.61 ±4.81）%;the cytotoxicities in A,B,C groups at E：T ratio = 40：1 were（67.7 ± 4.77）%,（51.51 ±443）%and（44.12 ± 5.62）%respectively.Meanwhile,the cytotoxicity in group A was significantly higher than that in group C at different E：T ratio（P〈0.05）,the percentage of cytotoxicity was（36.56 ± 3.69）%vs（10.85 ±2.09）%,（47.83 ±5.52）%vs（29.61 ±4.81）%,（67.7 ±4.77）%vs（44.12 ±5.62）%,respectively.Conclusion：CFSE/Annectin-V/7-AAD is able to clearly show human NK cell cytotoxicity against human tumors.Moreover,this staining technique also allows to distinguish different stages of cytotoxic killing as early and late apoptotic phase.

关 键 词：流式细胞技术 细胞毒作用 NK细胞 CFSE Annexin-V/7-AAD 

分 类 号：R446.6[医药卫生—诊断学]