外源性ATP对牙龈成纤维细胞NLRP3炎症小体活化的影响

doi:10.19439/j.sjos.2018.04.002

上海口腔医学 ›› 2018, Vol. 27 ›› Issue (4): 342-348.doi: 10.19439/j.sjos.2018.04.002

外源性ATP对牙龈成纤维细胞NLRP3炎症小体活化的影响

赖欣添, 夏一如, 谢玉峰^*, 束蓉^*

上海交通大学医学院附属第九人民医院·口腔医学院牙周病科,国家口腔疾病临床研究中心, 上海市口腔医学重点实验室,上海市口腔医学研究所,上海 200011

收稿日期:2018-01-11 修回日期:2018-03-29 出版日期:2018-08-25 发布日期:2018-10-09
通讯作者: 束蓉,E-mail：shurong123@hotmail.com;谢玉峰,E-mail：yufengxie@hotmail.com。^*共同通信作者
作者简介:赖欣添（1992-）,女,硕士研究生,E-mail: xintianlai005@163.com
基金资助:
国家自然科学基金（81570977); 上海市科研计划项目（18ZR1422400）

Effect of exogenous ATP on NLRP3 inflammasome activation in gingival fibroblasts cells

LAI Xin-tian, XIA Yi-ru, XIE Yu-feng, SHU Rong

Department of Periodontology, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology. Shanghai 200011, China;

Received:2018-01-11 Revised:2018-03-29 Online:2018-08-25 Published:2018-10-09

摘要/Abstract

摘要： 目的: 观察外源性三磷酸腺苷（ATP）对牙龈卟啉单胞菌(P.gingivalis)和热灭活牙龈卟啉单胞菌(HP.gingivalis)感染的人牙龈成纤维细胞NLRP3 炎症小体（inflammasome）的活化以及下游因子IL-1β分泌的影响。方法: 组织块法体外培养人牙龈成纤维细胞（hGFs）获取原代细胞,经5 mmol/L ATP预处理,用100 MOI P.gingivalis、100 MOI HP.gingivalis体外刺激hGFs,实时定量PCR检测NLRP3、ASC、Caspase-1、IL-1β的基因表达;Western免疫印迹技术检测细胞内NLRP3、Caspase-1和IL-1β蛋白的表达;ELISA法检测白细胞介素1β（IL-1β）的分泌。采用Graphpad prism 6软件包对数据进行t检验或单因素方差分析。结果: 与对照组相比,P.gingivalis 下调NLRP3 、ASC mRNA,上调IL-1β基因表达,下调NLRP3 、IL-1β胞内蛋白水平。HP.gingivalis 诱导NLRP3 、IL-1β、ASC基因和胞内蛋白的表达,P.gingivalis或者 HP.gingivalis单独刺激对Caspase-1 mRNA水平及IL-1β分泌均无影响。ATP/P.gingivalis或ATP/HP.gingivalis 共刺激均明显上调NLRP3、ASC、Caspase-1和IL-1β基因及胞内蛋白水平,增加上清液中IL-1β的分泌水平。结论: 外源性ATP可调控牙周主要致病菌P.gingivalis感染的人牙龈成纤维细胞 NLRP3炎症小体的激活,介导炎症因子IL-1β的成熟与分泌。

关键词: 牙龈卟啉单胞菌, 牙龈成纤维细胞, NLRP3炎症小体, 三磷酸腺苷, 炎症因子

Abstract: PURPOSE: To investigate exogenous ATP-dependent activation of NLRP3 inflammasome and interleukin-1β ( IL-1β) secretion in P.gingivalis infected and heat-killed P.gingivalis induced gingival fibroblasts cells ( hGFs) in vitro. METHODS: Gingival tissues were obtained from healthy patients and hGFs were cultured in vitro with tissue block method to harvest primary cells. HGFs was simulated by being treated with 100 MOI live P.gingivalis or 100 MOI heat-killed P.gingivalis (HP.gingivalis) after 5 mmol/L ATP pre-treatment. Real-time PCR was carried out to assess mRNA expression of NLRP3, ASC, caspase-1 and IL-1β. The protein level of NLRP3 , caspase-1 and IL-1β was evaluated by Western blot. IL-1β secretion was measured using ELISA. Statistical analysis was performed using Graphpad prism 6 statistical package and the measurement data were analyzed by t test or one-way ANOVA. RESULTS: Compared with the control group, P.gingivalis downregulated NLRP3 mRNA and ASC mRNA while upregulated IL-1β mRNA. Moreover, the protein expression of NLRP3 and IL-1β was decreased. The gene and protein expression of NLRP3, ASC and IL-1β was induced by HP.gingivalis, while caspase-1mRNA and IL-1βsecretion was free from P.gingivalis or HP.gingivalis stimulus. All those genes as well as intracellular protein expression and IL-1βsecretion were significantly potentiated with ATP/P.gingivalis or ATP/HP.gingivalis stimuli in hGFs. CONCLUSIONS: Exogenous ATP may be a potential stimulus signal in favour of NLRP3 inflammasome activation of hGFs and mediated inflammatory factor IL-1β secretion.

Key words: Porphyromonas gingivalis, hGFs, NLRP3 inflammasome, ATP, Inflammatory factor

中图分类号:

R781.4

赖欣添, 夏一如, 谢玉峰, 束蓉. 外源性ATP对牙龈成纤维细胞NLRP3炎症小体活化的影响[J]. 上海口腔医学, 2018, 27(4): 342-348.

LAI Xin-tian, XIA Yi-ru, XIE Yu-feng, SHU Rong. Effect of exogenous ATP on NLRP3 inflammasome activation in gingival fibroblasts cells[J]. Shanghai Journal of Stomatology, 2018, 27(4): 342-348.

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外源性ATP对牙龈成纤维细胞NLRP3炎症小体活化的影响

Effect of exogenous ATP on NLRP3 inflammasome activation in gingival fibroblasts cells

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