3种不同抗凝剂对外周血培养的NK细胞功能影响  

Influence of NK cells with three different anticoagulants treatments in peripheral blood culture

作　　者：陈玲[1] 吕小婷[1] 徐涛[1] 周忠海[1] 姚仁南[2] CHEN Ling;Lti Xiao-ting;XU Tao;ZHOU Zhong-hai;YAO Ren-nan(Department of Central Laboratory;Department of Blood Transfusion,the 97th Hospital of PLA,Xuzhou 221004,Jiangsu,China)

机构地区：[1]中国人民解放军第九七医院实验科,江苏徐州221004 [2]中国人民解放军第九七医院输血科,江苏徐州221004

出　　处：《生物医学工程与临床》2018年第5期572-577,共6页Biomedical Engineering and Clinical Medicine

基　　金：南京军区医学科技创新重点课题(14ZD17)

摘　　要：目的探讨外周血经3种不同抗凝剂处理后培养的自然杀伤(NK)细胞的增殖率和杀伤功能的影响研究。方法采集10例年龄(35±5)岁健康志愿者15 m L外周血,分别置于含乙二胺四乙酸二钾(EDTA-K2,EDTA组)、细胞保存液(细胞保存液组)和肝素钠(肝素钠组)的抗凝管中。用淋巴细胞分离液收集单个核细胞,在NK细胞培养液中培养单个核细胞12 d,并观察细胞生长情况。在第4、6、8、10、12天时,收集细胞用CCK8法检测NK细胞的增殖情况。在细胞增殖最明显的第12天收集细胞,通过流式细胞仪检测各组NK细胞的颗粒酶、穿孔素和CD107a。台盼蓝染色检测细胞活性。通过CCK8法检测各组对肿瘤细胞株K562杀伤活性。结果通过细胞增殖发现,EDTA组NK细胞增殖不明显,肝素钠组和细胞保存液组增殖明显。第12天时检测发现,肝素钠组增殖倍数为128.15±6.38,细胞保存液组增殖倍数为107.51±5.70;肝素钠组优于细胞保存液组(t=6.20,P=0.00)。肝素钠组NK细胞纯度表达为(84.49±2.35)%,也明显高于细胞保存液组NK细胞表达[(77.63±1.37)%]。经台盼蓝染色检测,肝素钠组和细胞保存液组细胞活性率均为100%。通过流式细胞仪检测发现,肝素钠组NK细胞颗粒酶B、穿孔素和CD107a的表达都明显高于细胞保存液组(t=12.76、17.76、5.67,P=0.00、0.00、0.00)。对肿瘤细胞株K562杀伤结果显示,肝素钠组NK细胞杀伤活性(50.12%±3.42%)明显高于细胞保存液组(41.88%±2.47%)(t=4.78,P=0.001)。结论肝素钠和细胞保存液均可用于NK细胞培养的血液抗凝,但肝素钠组NK细胞的杀伤功能明显优于细胞保存液组。Objective To investigate the effects of three kinds of anticoagulants of peripheral blood samples on the proliferation and killing activity of culture natural killer（NK） cells. Methods Ten healthy volunteers aged（35 ± 5） years old were enrolled, the 15 m L peripheral blood was collected and added with heparin sodium（heparin sodium group）, cell preservation liq-uid（sodium citrate, cell preservation liquid group） and ethylene diamine tetraacetic acid-dipotassium salt（EDTA-K2, EDTA group）. Mononuclear cells were collected by lymphocyte separation medium and cultivated in NK cells culture medium for 12 days and observed for cell growth. The proliferation of NK cells were determined by cell counting kit（CCK8） assay at the 4, 6,8, 10, 12 day. The expression of perforin, granzyme and CD107 a were detected by flow cytometry on the 12 th day（cell prolifer-ation was obvious）. The cell activity was detected by trypan blue staining, and CCK8 assay was used to measure killing activity for tumor cell line K-562 cells. Results The results showed that the proliferation of NK cells in EDTA group was not obvious, while the proliferations in heparin sodium group and cell preservation liquid group were significant. On 12 th day, the pro-liferation in heparin sodium group was 128.15 ± 6.38, in cell preservation liquid group was 107.51 ± 5.70, and the proliferation in heparin sodium group was significantly better than that in cell preservation liquid group（ t = 6.20, P = 0.00）. The purity expression of NK cells in heparin sodium group was（84.49 ± 2.35） %, which was also significantly higher than that in cell preservation liquid group[（77.63 ± 1.37） %]. The activity rates of both heparin sodium group and cell preservation liquid group stained with trypan blue solution were 100 %. The heparin sodium group was expressed higher in perforin, granzyme and CD107 a of NK cells than that of cell preservation liquid group（t = 12.76, 17.76, 5.67, P = 0.00, 0.00, 0.00）. The killing activity on tumo

关 键 词：抗凝剂 NK细胞 细胞增殖 杀伤功能 乙二胺四乙酸二钾(EDTA-K2) 细胞保存液 肝素钠 

分 类 号：R446.6[医药卫生—诊断学]