CFSE标记结合流式细胞术在研究NK细胞分裂与杀伤功能中的应用

Establishment of CFSE labeling method coupling with flow cytometry used in the study of human NK cell function

目的建立并优化CFSE标记结合流式细胞术检测人NK细胞分裂、增殖和杀伤功能的方法。方法利用CFSE标记磁珠纯化的人NK细胞,加或不加IL-2进行培养。分别在第3、5、7天收集细胞,流式细胞术对NK细胞的分裂增殖进行检测;利用IL-2活化NK细胞作为效应细胞,与靶细胞(K562细胞)按不同效靶比在37℃5%CO_2培养箱共孵育4 h加入碘化丙啶(PI),流式细胞术检测CFSE^+PI^+K562细胞(死亡的靶细胞)的百分率。结果CFSE标记的NK细胞在IL-2的作用下第5天即可观察到细胞增殖,至第7天80%以上的NK细胞均发生增殖,分裂1～6次。在对NK细胞杀伤功能的研究中,发现IL-2刺激后NK细胞的杀伤功显著增强。此外,当IL-2或IL-12活化的NK细胞作用于不同的靶细胞时,均可有效地杀伤靶细胞。结论以CFSE标记技术结合流式细胞术可在单细胞水平客观精确地对NK细胞的增殖、分裂和杀伤功能进行分析。本方法灵敏度高、操作简便易行、重复性好,对其他淋巴细胞亚群功能的研究具有重要的借鉴意义。

To establish an optimal CFSE labeling methods for the study of NK cell functions.We purified human NK cells and labeled the cells with CFSE;the labeled NK cells were cultured in presence or absence of interleukin 2（IL-2） at 37℃5%CO_2 incubator, and then harvested at day 3,5 and 7 for determining the proliferation and divisions of activated NK cells by using flow cytometry.For analysis of cytolytic activity,the NK cells were incubated with IL-2 and co-cultured with target cells for 4 h at 37℃5%CO_2 incubator,and then propidium iodide（PI）was added into the cultures.CFSE^＋PI^＋ cells were determined as the dead cells by flow cytometry.The proliferation of IL-2-activated NK cells could be detected at day 5,and about 80%of NK cells were on proliferation at day 7 and divided from one to six times.On the other hand,the cytolytic effects of NK cells on CFSE-labeled different target cells were enhanced after activation with IL-2 and IL-12.The results showed that the fluorescent label ester CFSE is a convenient method to tag proliferating cells,which allows discrimination of successive rounds of cell division.Based on target cells labeled with CFSE and subsequent DNA labeled with PI,the cytotoxicity of NK cells could be detected in a single cell level.This study might provide valuable information on the function study methods for NK cells as well as other lymphocyte subsets.