粉尘螨诱导结膜上皮细胞损伤促进中性粒细胞迁移与中性粒细胞胞外陷阱形成

中国血吸虫病防治杂志 ›› 2023, Vol. 35 ›› Issue (3): 271-278.

粉尘螨诱导结膜上皮细胞损伤促进中性粒细胞迁移与中性粒细胞胞外陷阱形成

吴美丽1，闫茹2，赵文君3*

1南京医科大学附属无锡人民医院临床研究中心（江苏无锡 214023）；2 江南大学附属儿童医院儿科实验室；3 南京医科大学附属无锡人民医院检验科（江苏无锡 214023）

出版日期:2023-06-25 发布日期:2023-07-05
作者简介:吴美丽，女，硕士，医师。研究方向：过敏性疾病
基金资助:
江苏省无锡市卫生健康委科研项目（Q202021)

Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation

WU Meili1, YAN Ru2, ZHAO Wenjun3*

1 Center of Clinical Research, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, Jiangsu 214023, China;2 Department of Pediatrics Laboratory, Affiliated Children’s Hospital of Jiangnan University, China; 3 Department of Clinical Laboratory, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, Jiangsu 214023, China

Online:2023-06-25 Published:2023-07-05

摘要/Abstract

摘要： 目的　探讨粉尘螨粗提浸液刺激人结膜上皮细胞损伤及诱导中性粒细胞迁移和中性粒细胞胞外陷阱（neutrophil extracellular traps，NETs）形成，从而引起过敏性结膜炎的发病机制。方法　采用500、1 000、2 000、4 000 ng/mL 粉尘螨粗提浸液刺激人结膜上皮细胞，采用实时荧光定量PCR（quantitative real⁃time PCR，qPCR）和酶联免疫吸附试验（enzyme⁃linked immunosorbent assay，ELISA）检测人结膜上皮细胞中白细胞介素6（interleukin⁃6，IL⁃6）、肿瘤坏死因子α（tumor necrosis factor⁃α，TNF⁃α）、γ干扰素（interferon⁃γ，IFN⁃γ）和IL⁃8表达水平。收集人结膜上皮细胞培养上清液，与中性粒细胞共培养，采用Transwell细胞迁移实验检测中性粒细胞迁移数量，采用免疫荧光染色检测NETs标志物髓过氧化物酶（myeloperoxidase，MPO）和瓜氨酸化组蛋白H3（citrullinated histone H3，CitH3）表达。采用佛波酯（phorbol 12⁃myristate 13⁃acetate，PMA）刺激中性粒细胞并收集NETs，应用NETs处理人结膜上皮细胞，采用流式细胞术检测细胞凋亡，采用ELISA法检测细胞培养上清中IL⁃6、TNF⁃α、IFN⁃γ和IL⁃8水平。结果　2 000、4 000 ng/mL粉尘螨粗提浸液处理上调人结膜上皮细胞中IL⁃6、TNF⁃α、IFN⁃γ和IL⁃8表达水平；经2 000、4 000 ng/mL粉尘螨粗提浸液处理后，人结膜上皮细胞培养上清可促进中性粒细胞迁移，诱导MPO和CitH3染色丝状物增加；增加的NETs进一步诱导人结膜上皮细胞凋亡和IL⁃6、TNF⁃α、IFN⁃γ和IL⁃8释放。结论　粉尘螨粗提浸液诱导人结膜上皮细胞损伤，从而促进中性粒细胞迁移与NETs形成，而NETs释放进一步加剧人结膜上皮细胞损伤。

关键词: 粉尘螨, 过敏性结膜炎, 人结膜上皮细胞, 中性粒细胞, 中性粒细胞胞外陷阱

Abstract: Objective　To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM). Methods　Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin⁃6 (IL⁃6), tumor necrosis factor⁃α (TNF⁃α), interferon⁃γ (IFN⁃γ) and IL⁃8 were detected using quantitative real⁃time PCR (qPCR) assay and enzyme⁃linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co⁃cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12⁃myristate 13⁃acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL⁃6, TNF⁃α, IFN⁃γ and IL⁃8 were measured in the cell culture supernatant using ELISA. Results　Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up⁃regulated IL⁃6, TNF⁃α, IFN⁃γ and IL⁃8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL⁃6, TNF⁃α, IFN⁃γ and IL⁃8 secretion in the culture supernatant of human conjunctival epithelial cells. Conclusions　CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

Key words: Dermatophagoides farinae, Allergic conjunctivitis, Human conjunctival epithelial cell, Neutrophil, Neutrophil extracellular traps

中图分类号:

R384.4

吴美丽, 闫茹, 赵文君. 粉尘螨诱导结膜上皮细胞损伤促进中性粒细胞迁移与中性粒细胞胞外陷阱形成[J]. 中国血吸虫病防治杂志, 2023, 35(3): 271-278.

WU Meili, YAN Ru, ZHAO Wenjun. Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation [J]. Chinese Journal of Schistosomiasis Control, 2023, 35(3): 271-278.

[1]	周小倩, 马杰, 王睿怡, 王若航, 吴毅强, 杨欣宴, 陈玉娟, 唐小牛, 孙恩涛. 基于高通量测序的粉尘螨体内细菌多样性研究 [J]. 中国血吸虫病防治杂志, 2022, 34(6): 630-.
[2]	湛孝东, 姚润, 汪梅青, 蒋峰, 郭伟, 汤非凡, 王澜, 李朝品. 粉尘螨各发育阶段外部形态扫描电镜观察 [J]. 中国血吸虫病防治杂志, 2022, 34(2): 179-.
[3]	李汶霖, 许志伟, 李淑凝, 沈海娥, 王洋, 李志敏, 张晨烁, 邢阳光, 沈鑫. 中性粒细胞胞外诱捕网在抗寄生虫免疫中的作用研究进展[J]. 中国血吸虫病防治杂志, 2021, 33(4): 434-.
[4]	杨华莹，朱晗婷，崔玉宝. 粉尘螨瘦素受体叠加转录蛋白样1基因克隆及序列分析[J]. 中国血吸虫病防治杂志, 2020, 32(3): 248-.
[5]	陈静，王康，南丹阳，钱文斯，胡燕，陈文捷，王少圣，孙恩涛. 芜湖市某高校学生宿舍床席螨类和昆虫孳生状况调查[J]. 中国血吸虫病防治杂志, 2016, 28(2): 151-155.
[6]	宋红玉, 段彬彬, 李朝品. ProDer f 1多肽疫苗免疫治疗粉螨性哮喘小鼠的效果[J]. 中国血吸虫病防治杂志, 2015, 27(5): 490-.
[7]	徐海丰, 祝海滨, 徐朋飞, 李朝品, 王克霞. 粉尘螨1类变应原重组融合表位免疫治疗小鼠哮喘的效果分析[J]. 中国血吸虫病防治杂志, 2015, 27(1): 49-.
[8]	徐海丰, 徐朋飞, 王克霞, 李朝品. 粉尘螨1类变应原T和B细胞表位嵌合基因的构建与表达[J]. 中国血吸虫病防治杂志, 2014, 26(4): 420-.
[9]	李娜, 李朝品, 刁吉东, 赵蓓蓓, 姜玉新. 粉尘螨3类变应原的T细胞表位预测及鉴定[J]. 中国血吸虫病防治杂志, 2014, 26(4): 415-.
[10]	李娜, 李朝品, 刁吉东, 赵蓓蓓, 陆维, 姜玉新. 粉尘螨3类变应原的B细胞线性表位预测及鉴定[J]. 中国血吸虫病防治杂志, 2014, 26(3): 296-.
[11]	许永良, 李洪军, 黄一心, 余传信, 王铁生, 吴中兴. 吡喹酮对血吸虫卵肉芽肿与肝纤维化作用的研究 II 吡喹酮对小鼠肺部血吸虫卵肉芽肿内细胞的影响[J]. 中国血吸虫病防治杂志, 2010, 22(2): 105-110.