Wnt5a对中性粒细胞胞外诱捕网形成的影响  

Effects of Wnt5a on the formation of neutrophil extracellular trap

作　　者：陆敏 卢伟[1,2,3] 杨勰 孙颖[1,2,3] LU Min;LU Wei;YANG Xie;SUN Ying(Department of Periodontology,the Affiliated Stomatological Hospital of Nanjing Medical University,Nanjing 210029,China;Jiangsu Key Laboratory of Oral Diseases of Nanjing Medical University,Nanjing 210029,China;Jiangsu Province Engineering Research Center of Stomatological Translational Medicine,Nanjing 210029,China)

机构地区：[1]南京医科大学附属口腔医院牙周科,江苏南京210029 [2]南京医科大学江苏省口腔疾病研究重点实验室,江苏南京210029 [3]江苏省口腔转化医学工程中心,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2021年第6期826-831,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81771075);江苏高校优势学科建设工程资助项目(2018-87);南京医科大学科技发展基金(2016NJMU052)。

摘　　要：目的:探讨Wnt5a对中性粒细胞胞外诱捕网(neutrophil extracellular trap,NET)形成的影响及可能的调控机制。方法:采用50 ng/mL Wnt5a刺激人中性粒细胞3 h,以1μg/mL牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)脂多糖(lipopolysaccharide,LPS)和1μg/mL大肠杆菌(Escherichia coli,E.coli)LPS作为阳性对照,采用免疫荧光技术观察NET的形成,Sytox Green荧光法对胞外DNA进行定量,采用流式细胞术检测细胞产生的活性氧(reactive oxygen species,ROS)水平,Western blot分析信号分子磷脂酰肌醇三激酶(phosphoinositide 3-kinase,PI3K)/Akt和丝裂原活化蛋白激酶1/2(mitogen-activated protein kinase1/2,MEK1/2)、细胞外信号调节蛋白激酶1/2(extracellular regulated protein kinases1/2,ERK1/2)表达水平的变化。结果:Wnt5a刺激人中性粒细胞后,可观察到NET的形成和胞外DNA数量的增加(P <0.05),ROS水平较刺激前明显增高(P <0.05),Western blot检测显示,PI3K/Akt和MEK1/2、RK1/2磷酸化水平增高(P <0.05)。结论:Wnt5a可能通过PI3K/Akt、MEK1/2、ERK1/2磷酸化,调控ROS生成,进而促进人中性粒细胞形成NET。Objective:This study aims to observe the effects and mechanisms of Wnt5 a on the formation of neutrophil extracellular trap(NET). Methods:Neutrophils were stimulated with 50 ng/mL Wnt5 a for 3 h,1 μg/mL Porphyromonas gingivalis(P.gingivalis)lipopolysaccharide(LPS)and 1 μg/mL Escherichia coli(E.coli)LPS were served as positive controls. Formation of NET was observed by immunofluorescence staining,and extracellular DNA was quantified by Sytox Green fluorescence staining. Levels of reactive oxygen species(ROS)were detected by flow cytometry,and activations of phosphatidylinositol 3-kinase(PI3 K)/Akt,mitogen-activated protein kinase 1/2(MEK1/2)and extracellular regulated protein kinases 1/2(ERK1/2)were explored by Western blot. Results:Compared with neutrophils without stimulations,Wnt5 a stimulation led to the formation of NET and the increased levels of extracellular DNA(P <0.05). In addition,levels of ROS and phosphorylations of PI3 K/Akt,MEK1/2 and ERK1/2 were significantly increased in the cells treated with Wnt5 a(P < 0.05). Conclusion:Wnt5 a might contribute to the formation of NET depending on the production of ROS and the phosphorylations of PI3 K/Akt,MEK1/2 and ERK1/2.

关 键 词：WNT5A 中性粒细胞胞外诱捕网 ROS PI3K/AKT MEK1/2-ERK1/2 

分 类 号：R392.12[医药卫生—免疫学]