一种新型稳定的流式细胞术检测NK细胞活性的方法

摘要

目的 建立一种新型稳定的流式细胞术检测人外周血NK细胞活性的方法.方法 通过慢病毒转染技术构建稳定表达增强型绿色荧光蛋白(EGFP)的K562靶细胞株,采用Annexin V-PE/7AAD凋亡染色标记,进行流式细胞术检测,经NK细胞杀伤作用后,靶细胞的凋亡比例即反映出NK细胞的杀伤活性.选取43名成年健康志愿者及37例成年噬血细胞综合征(HPS)确诊患者进行NK细胞活性的检测,比较两者之间NK细胞活性的差异.结果 健康志愿者与HPS确诊患者性别比例(男∶女为21∶22比21∶16,)及年龄[(30.53±7.13)岁比(33.24±10.78)岁]差异均无统计学意义(P=0.479,P=0.198),HPS患者NK细胞活性低于健康志愿者[(11.79±2.73)％比(21.08±4.26)％,P＜ 0.001].结论 该检测方法快速、准确、稳定,适宜推广,可为NK细胞杀伤活性的检测提供一种新的方法.%Objective To establish a new stable flow cytometric detection method for natural killer (NK) cell activity in human peripheral blood.Methods K562 target cell line with an enhanced green fluorescent protein (EGFP) stable expression was established by lentivirus-mediated transfection method.The AnnexinV-PE/7AAD double staining markers were used for apoptosis labeling,followed by a flow cytometric test.The percentage of apoptotic target cells reflected cytotoxic activity of NK cells.To verify this novel method,43 adult healthy volunteers and 37 adult hemophagocytic syndrome (HPS) patients were selected for comparing the difference of NK cell activity between the two groups.Results There were no significant differences in gender (M/F 21∶22 vs.21∶16,P=0.479) and age [(30.53±7.13) y vs.(33.24±10.78) y,P=0.198] between 43 healthy volunteers and 37 HPS patients,whereas the cytotoxic function of NK cells in HPS patients was significantly lower than that in health persons [(11.79±2.73) ％ vs.(21.08±4.26) ％,P ＜ 0.001].Conclusion This detection method is rapid,accurate and stable,and it can be easily accomplished in most clinical laboratories,which provides a new approach to determine the cytotoxic activity of NK cells.