利用流式细胞仪检测人外周血NK细胞毒性方法的建立

目的利用流式细胞仪(FACS)技术检测人外周血NK细胞的毒性。方法首先将pEGFP-N1质粒转染人NK细胞的天然靶细胞K562,经过G418筛选后进一步单克隆化,得到稳定、均一表达绿色荧光蛋白的K562细胞。取对数期的K562-EGFP细胞与人外周血单个核细胞分别按5∶1、10∶1、20∶1、40∶1的比例混合,分别孵育0.5、1、2、4 h后用碘化丙啶(PI)标记,用流式细胞仪分析呈红、绿色荧光的细胞数,最后计算NK细胞的杀伤效率。结果0.5、1、2、4 h均能得到明显的杀伤效果,而且以2h的杀伤率最高。此时杀伤率与传统乳酸脱氢酶法(LDH)具有显著相关性(γ=0.997,P=0.003),而且显示出更强的敏感性。结论利用流式细胞仪检测NK细胞毒活性的方法可作为传统51Cr释放法、LDH法的一个补充,而且具有经济、快速和敏感的特点。

MEASURING HUMAN PERIPHERAL NK CELL CYTOTOXICITY WITH FLOW CYTOMETRY

Objective To establish a method of detecting human peripheral NK cell cytotoxicity with flow cytometry.Methods Enhanced green fluorescent protein(EGFP) was stably expressed in human erythroleukaemia K562 cells(EGFP-K562) and used as target cells.PBMCs were mixed with log-phase K562-EGFP cells in a series of effector and target ratio of 5∶1,10∶1,20∶1,40∶1.After 0.5,1,2,4 hour(s) of incubation,cells were labeled with propidium iodide(PI) and showed dual(green-red) fluorescent.Results Although the kinetic study demonstrated that all of the incubations got the cytolysis,the 2 hour incubation was the optional time.This technique strongly correlated with the traditional lactate dehydrogenase release assay at the correlation coefficients of 0.997 and 0.961 at p-value of 0.003 and 0.04 for 2 and 4 h incubation times respectively.In addition,our method showed a higher sensitivity.Conclusions This is a simple,reliable,safe and high sensitive method to measure NK cytotoxicity with flow cytometry.