外周血中性粒细胞和口腔中性粒细胞免疫炎症反应的差异比较

口腔生物医学 ›› 2023, Vol. 14 ›› Issue (2): 80-86.

外周血中性粒细胞和口腔中性粒细胞免疫炎症反应的差异比较

朱沁,高岚青,仝悦,孙颖*

南京医科大学附属口腔医院牙周病科，江苏省口腔疾病研究重点实验室，江苏省口腔转化医学工程研究中心

收稿日期:2023-02-02 修回日期:2023-04-19 出版日期:2023-06-25 发布日期:2023-07-31
通讯作者: 孙颖 E-mail:ebolasun@163.com
基金资助:
国家自然科学基金;江苏省科教能力提升工程—江苏省研究型医院;江苏省医学创新中心;南京医科大学科技发展基金

Differences in inflammatory responses between oral polymorphonuclear neutrophil and circulating polymorphonuclear neutrophil

Received:2023-02-02 Revised:2023-04-19 Online:2023-06-25 Published:2023-07-31

摘要/Abstract

摘要：

目的：比较口腔中性粒细胞（oPMN）和外周血中性粒细胞（cPMN）免疫炎症反应的差异，探讨oPMN维持牙周健康的作用。方法：常规密度梯度离心法分离获得健康人外周静脉血来源的cPMN，离心过滤法分离获得健康人口腔含漱液来源的oPMN。采用1 μg/mL牙龈卟啉单胞菌（P. gingivalis）脂多糖（LPS）刺激，并以大肠杆菌（E. coli）LPS作为阳性对照，流式细胞术检测凋亡水平变化；Transwell实验比较白介素（IL）-8刺激前后两种细胞趋化能力的差异；ELISA检测IL-1β、IL-8和IL-10表达水平变化；流式细胞术检测两种细胞对P. gingivalis或E. coli的吞噬作用；免疫荧光实验观察中性粒细胞胞外诱捕网（NETs）的形成，Sytox Green法对胞外DNA进行定量；Western blot检测丝裂原活化蛋白激酶1/2（MEK1/2）、细胞外信号调节蛋白激酶1/2（ERK1/2）和核转录因子-κB（NF-κB）P65磷酸化水平的变化。结果：两种LPS刺激后，与cPMN相比，oPMN凋亡增多（P<0.05）。IL-8刺激后，两种细胞趋化能力未见明显差异（P>0.05）。两种LPS刺激后，oPMN较cPMN的IL-1β、IL-8和IL-10表达水平更高（P<0.01）。oPMN吞噬P. gingivalis和E. coli的能力强于cPMN（P<0.05），形成的NETs更多，胞外DNA水平、MEK1/2、ERK1/2和NF-κB P65磷酸化水平均较cPMN更高（P<0.05）。结论：oPMN可能是具有过度活跃表型的中性粒细胞，其吞噬、分泌细胞因子和形成NETs的能力较cPMN增强，可能与其MEK1/2、ERK1/2和NF-κB磷酸化水平增高有关。

Abstract: Objective: To explore the differences in inflammatory responses between oral polymorphonuclear neutrophil (oPMN) and circulating polymorphonuclear neutrophil (cPMN), and the roles of oPMN in maintaining periodontal health. Methods：cPMN was obtained from peripheral venous blood of healthy individuals by density gradient centrifugation, while oPMN was collected from oral rinses of healthy subjects by centrifugal filtration. Both of PMNs were stimulated with 1μg/ml Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and Escherichia coli (E. coli) LPS was employed as a positive control. Apoptosis levels and phagocytosis of P. gingivalis or E. coli by two PMNs were assessed by flow cytometry. Moreover, after IL-8 stimulation, chemotactic responses of the cells were determined by a transwell migration assay. Production of interleukin (IL)-1β, IL-8 and IL-10 were analyzed by ELISA. Formation of neutrophil extracellular traps (NETs) was observed by immunofluorescence staining and levels of extracellular DNA were quantified by Sytox Green fluorescence staining. Furthermore, phosphorylation of mitogen?activated protein kinase1/2 (MEK1/2), extracellular regulated protein kinases1/2 (ERK1/2) and nuclear factor kappa-B (NF-κB) were measured by Western blot. Results：After P. gingivalis/E. coli LPS stimulation, higher levels of apoptosis in oPMN were disclosed compared with cPMN (p<0.05). No significant difference was confirmed in chemotactic ability of the two PMNs after IL-8 stimulation (p>0.05). Expression levels of IL-1β, IL-8 and IL-10 in oPMN were higher compared with cPMN (p<0.05) and more quantities of P. gingivalis or E. coli were phagocytized by oPMN than cPMN (p<0.05). Moreover, more quantities of NETs, higher levels of extracellular DNA, and greater phosphorylation levels of MEK1/2, ERK1/2 and NF-κB P65 were revealed in oPMN compared with cPMN (p<0.05). Conclusion：oPMN might be an overactive phenotype of neutrophil with increased capacities of phagocytosis, NET formation and cytokine secretion compared to cPMN, which might be associated with the increased phosphorylation of MEK1/2, ERK1/2 and NF-κB.

朱沁高岚青仝悦孙颖. 外周血中性粒细胞和口腔中性粒细胞免疫炎症反应的差异比较[J]. 口腔生物医学, 2023, 14(2): 80-86.

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