不同抗凝剂对体外中性粒细胞吞噬、活化和凋亡功能的影响

Effects of different anticoagulants on phagocytosis,activation and apoptosis of in vitro neutrophil function

目的评估不同抗凝剂对中性粒细胞吞噬、活化和凋亡功能的影响。方法选取2020年1月—2020年6月河南省人民医院体检健康志愿者30名,随机平均分为3组。分别采用肝素钠抗凝管采集外周血3 mL(肝素钠组),用乙二胺四乙酸二钾(EDTA-K_(2))抗凝管采集外周血2 mL(EDTA-K_(2)组),用柠檬酸钠抗凝管采集外周血2 mL(柠檬酸钠组)。采用流式细胞术检测中性粒细胞吞噬OVA-FITC的平均荧光强度,判断中性粒细胞吞噬能力;检测中性粒细胞表面活化标志物CD62L、CD11B的表达,判断中性粒细胞活化情况;基于7AAD、Annexin-V分析中性粒细胞凋亡情况。结果EDTA-K_(2)组中性粒细胞吞噬OVA-FITC的平均荧光强度低于肝素钠组、柠檬酸钠组(P<0.05)。EDTA-K_(2)组中性粒细胞CD11B表达低于肝素钠组和柠檬酸钠组(P<0.01、P<0.05)。与肝素钠组相比,EDTA-K_(2)组和柠檬酸钠组中性粒细胞凋亡增加(P<0.01、P<0.05)。结论不同抗凝剂对体外中性粒细胞的吞噬、活化和凋亡功能影响程度不同,临床应选择合适的抗凝剂,以准确分析中性粒细胞的吞噬、活化和凋亡功能。

Objective To evaluate the effects of different anticoagulants on phagocytosis,activation and apoptosis of in vitro neutrophil function.Methods Totally,30 healthy subjects who underwent physical examination in Henan Provincial People's Hospital from January 2020 to June 2020 were enrolled,and they were classified into 3 groups randomly(10 cases for each group).The 3 mL peripheral blood was collected with heparin sodium anticoagulant tube(heparin sodium group),2 mL peripheral blood was collected with ethylenediaminetetraacetic acid(EDTA)-K_(2) anticoagulant tube(EDTA-K_(2) group),and 2 mL peripheral blood was collected with sodium citrate anticoagulant tube(sodium citrate group).The average fluorescence intensity of OVA-FITC was determined by flow cytometry to determine the phagocytosis ability of neutrophils.The expressions of neutrophil surface activation markers,CD62L and CD11B,were determined to assess the activation of neutrophils.7AAD and Annexin-V were added to determine the apoptosis of neutrophils.Results The average fluorescence intensity of OVA-FITC phagocytosis neutrophils in EDTA-K_(2) group was lower than those in heparin sodium and sodium citrate groups(P<0.05).CD11B neutrophils in EDTA-K_(2) group were lower than those in heparin sodium and sodium citrate groups(P<0.01,P<0.05).Compared with heparin sodium group,neutrophil apoptosis was increased in EDTA-K_(2) and sodium citrate groups(P<0.01,P<0.05).Conclusions Different anticoagulants can affect the phagocytosis,activation and apoptosis of neutrophils in vitro,but each anticoagulant has different effects.Therefore,appropriate anticoagulant must be selected to accurately determine the phagocytosis,activation and apoptosis of neutrophils.