Wnt5a对中性粒细胞胞外诱捕网形成的影响
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1.南京医科大学口腔疾病研究江苏省重点实验室;2. 南京医科大学附属口腔医院 牙周科

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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)


Effects of Wnt5a on the Formation of Neutrophil Extracellular Traps
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National Natural Science Foundation of China through project 81771075 and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (2018-87),Science and Technology Development Fund of Nanjing Medical University (2016NJMU052)

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    摘要:

    目的:探讨Wnt5a对中性粒细胞胞外诱捕网(neutrophil extracellular taps,NETs)形成的影响及可能的调控机制。方法:采用50ng/ml Wnt5a刺激人中性粒细胞3h,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NETs的形成,Sytox Green荧光法对胞外DNA进行定量,采用流式细胞技术检测细胞产生的活性氧(reactive oxygen species,ROS)水平,western blot技术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NETs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 / ERK1/2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NETs。

    Abstract:

    Objective: To observe the effects and mechanisms of Wnt5a on the formation of neutrophil extracellular traps (NETs). Methods: Neutrophils were stimulated with 50ng/ml Wnt5a for 3h, and 1μg/ml Porphyromonas gingivalis (P.gingivalis) lipopolysaccharide (LPS) and 1μg/ml Escherichia coli (E.coli) LPS were served as positive controls. Formation of NETs 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 (PI3K)/Akt, mitogen-activated protein kinase 1/2 (MEK1/2) / extracellular regulated protein kinases 1/2 (ERK1/2) were explored by western blot. Results: Compared with neutrophils without stimulations, Wnt5a stimulation led to the formation of NETs and the increased levels of extracellular DNA (p<0.05). In addition, levels of ROS and phosphorylations of PI3K / Akt and MEK1/2 / ERK1/2 were significantly increased in the cells treated with Wnt5a (p<0.05). Conclusion: Wnt5a might contribute to the formation of NETs depending on the production of ROS and the phosphorylations of PI3K/Akt and MEK1/2 / ERK1/2.

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  • 收稿日期:2020-05-31
  • 最后修改日期:2020-12-05
  • 录用日期:2021-06-21
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