细胞因子样蛋白1对脓毒症小鼠中性粒细胞的作用  

Cytokine-like protein 1 enhances the pro-inflammatory functions of neutrophils in septic mice

作　　者：李纾[1] 朱凤雪[1] 蒋丽蕾 薛海岩 安友仲[1] Li Shu;Zhu Fengxue;Jiang Lilei;Xue Haiyan;An Youzhong(Department of Critical Care Medicine,Trauma Centre,Peking University People's Hospital,Beijing 100044,China)

机构地区：[1]北京大学人民医院重症医学科,创伤救治中心,北京100044

出　　处：《中华急诊医学杂志》2020年第2期199-203,共5页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金(81641089)。

摘　　要：目的研究细胞因子样蛋白1(cytokine-like protein 1,CYTL1)在脓毒症早期天然免疫反应阶段,对中性粒细胞促炎功能的影响。方法利用C57BL/6小鼠,随机数表法随机分为脓毒症组(盲肠结扎穿孔法制备)和对照组(实施假手术),每组6-12只。术后8h后分离小鼠外周血中性粒细胞,用CYTL1重组蛋白作用于细胞。Boyden趋化小室检测CYTL1对细胞的趋化活性;荧光素标记大肠杆菌吞噬试剂盒检测吞噬功能;荧光探针标记后流式细胞术检测氧自由基释放。统计学分析使用SPSS 20.0统计分析软件,符合正态分布的计量资料采用均数士标准差(Mean±SD)表示,组间比较采用成组r检验;计数资料采用率表示,组间比较采用卡方检验。以P<0.05为差异有统计学意义。结果与对照组相比,CYTL1对脓毒症小鼠中性粒细胞有较强的趋化活性[(10.0±2.0)vs(66.3±4.0),r=-21.6,P<0.0001]。与其他趋化因子相比,CYTL1趋化活性较“中间型”趋化因子白细胞介素-8强[(66.3±4.0)vs(21.7±6.5),t=10.1,P=0.001];与“终点型”趋化因子fMLF相比差别不明显[(66.3±4.0)vs(86.0±13.5),t=-2.4,P=0.073]。流式细胞术及共聚焦激光显微镜检测结果显示,与脓毒症组相比,CYTL1蛋白能促进脓毒症小鼠中性粒细胞吞噬大肠杆菌[(7.35±1.66)vs(2.84±0.62),t=4.4,P=0.012],并增加细胞释放氧自由基[(84340.1±5353.5)vs(351018.7±72291.7),t=6.4,P=0.003]。结论在脓毒症天然免疫反应阶段,CYTL1对小鼠中性粒细胞有较强的趋化活性,且能促进细胞吞噬能力及氧自由基释放,提示该因子可能在疾病早期有促进炎症反应的作用。Objectives To analyze the effect of cytokine-like protein 1(CYTL1)on the proinflammatory functions of neutrophils in septic mice.Methods C57BL/6 mice were randomly(random number)divided into the sepsis group and control group,with 6-12 mice in each group.A septic mouse model was established by the procedure of cecal ligation and puncture(CLP).Neutrophils were isolated from peripheral venous blood 8 h after the procedures according to the density gradient centrifugation method,and the neutrophils were treated with CYTL1 recombinant protein.The Boyden chemotaxis assays were used to detect the activity of CYTL1.fMLF and interleukin-8 were used as positive controls.Phagocytosis was determined by confocal microscopy or on a FACSVerse.Reactive oxygen species generation in neutrophils were monitored with the commercial CellROX Green fluorescent probe.Results Compared with the control group,CYTL1 showed strong chemotactic activity on neutrophils of septic mice[(10.0±2)vs(66.3±4),t=-21.6,P<0.0001].CYTL1 has stronger chemotactic activity than IL-8[(66.3±4.0)vs(21.7±6.5),t=10.1,P=0.001].But the chemotactic activity of fMLF and CYTL1 changed little on neutrophils of septic mice[(66.3±4.0)vs(86.0±13.5),t=-2.4,P=0.073].CYTL1 could augment the uptake of E.coli by neutrophils compared with the sepsis group[(7.35±1.66)vs(2.84±0.62),t=4.4,P=0.012].The number of E.coli particles swallowed intracellular by a single cell significantly increased upon the stimulation of CYTL1.CYTL1 could also enhance the intracellular reactive oxygen species production of neutrophils of septic mice[(84340.1±5353.5)vs(351018.7±72291.7),t=6.4,P=0.003].Conclusions CYTL1 can prompt the pro-inflammatory functions of neutrophils in septic mice.In the early phase of bacterial infection,this protein may play an important role in regulating the inflammation.

关 键 词：细胞因子样蛋白1 中性粒细胞 趋化活性 吞噬功能 氧活性物质 脓毒症 天然免疫反应 炎症反应 

分 类 号：R459.7[医药卫生—急诊医学]