Egr-1对机械力诱导人牙周膜细胞炎症反应的作用及机制研究

摘要/Abstract

摘要： 目的研究早期生长转录因子（early growth responsive gene-1,Egr-1）对机械应力（mechanical stress,MS）介导人牙周膜细胞（human periodontal membrane cells,hPDLs）炎症因子分泌和表达的影响及可能机制。方法以实时定量PCR和ELISA法检测MS加载不同时间（6、12、24和48 h）和不同形变率（3%、6%、12%和15%）时炎症因子IL-1β、IL-6、IL-8和IL-11的分泌和表达水平。MS对Egr-1表达的影响采用实时定量PCR和Western印迹法检测,沉默Egr-1对炎症因子的作用采用实时定量PCR和ELISA检测。应用Western印迹法检测Egr-1下调对PTEN/PI3K/Akt信号通路相关蛋白表达的影响。进一步采用20 μmol/L PI3K/Akt抑制剂LY294002预处理hPDLs 30 min,研究Egr-1沉默在MS介导炎症因子表达中的可能机制。采用SPSS 11.0软件包对数据进行统计学分析。结果IL-1β、IL-6、IL-8和IL-11的分泌和mRNA表达随着作用时间和形变率的增加而升高, 12%的MS作用24 h后,其分泌程度和表达水平最高。12%的MS加载24 h显著上调Egr-1表达。沉默Egr-1显著抑制MS诱导炎症因子表达。Egr-1沉默下调PTEN表达,上调p-PI3K和p-Akt蛋白表达水平,LY294002预处理可部分阻断Egr-1沉默对炎症因子分泌及表达的抑制作用。结论沉默Egr-1通过PETN/PI3K/Akt信号通路抑制MS诱导的炎症因子分泌和表达。

关键词: 早期生长转录因子, 机械应力, 人牙周膜细胞, 炎症因子

Abstract: PURPOSE: To investigate the effects and mechanisms of silencing Egr-1 on MS-mediated secretion and expression of inflammatory cytokines in hPDLs. METHODS: The secretion and expression of IL-1β, IL-6, IL-8 and IL-11 were detected using ELISA and RT-PCR when cells were cultured with or without various duration (6, 12, 24 and 48 h) and force (3%, 6%, 12%, 15%) of mechanica stress (MS). RT-PCR and Western blotting were used to evaluate the expression of Egr-1 under a 12% MS for 24 h. The roles of silencing Egr-1 in MS-mediated expression of inflammatory cytokines were determined using ELISA and RT-PCR. The protein levels of PTEN/PI3K/Akt signaling pathway were determined using Western blotting. Moreover, cells were pretreated with 20 μmol/L LY294002 for 30 min, in order to study the mechanisms of Egr-1 in MS-mediated secretion and expression of inflammatory cytokines. Statistical analysis was performed using SPSS 11.0 software package. RESULTS: The secretion and expression of IL-1β, IL-6, IL-8 and IL-11 were increased gradually with the time under a MS force of 12% in hPDLs, and peaked in cells after exposure to MS for 24 h. The mRNA and protein levels of Egr-1 were elevated in hPDLs after exposure to 12% MS for 24 h. Moreover, depletion of Egr-1 inhibited MS-mediated secretion and expression of inflammatory cytokines. Knockdown of Egr-1 reduced the protein level of PTEN, and increased the protein expression of p-PI3K and p-Akt in hPDLs. LY294002 blocked partially the inhibitory roles of Egr-1 in the secretion and expression of inflammatory cytokines. CONCLUSIONS: Depletion of Egr-1 suppressed the secretion and expression of inflammatory cytokines induced by MS through PTEN/PI3K/Akt pathway.

Key words: Early growth responsive gene-1, Mechanical stress, Human periodontal membrane cells, Inflammatory cytokines

中图分类号:

R781.4

呼海燕, 高志彪. Egr-1对机械力诱导人牙周膜细胞炎症反应的作用及机制研究[J]. 上海口腔医学, 2016, 25(5): 553-559.

HU Hai-yan, GAO Zhi-biao. Effects and mechanisms of Egr-1 on inflammatory responses induced by mechanical stress in human periodontal ligament cells[J]. Shanghai Journal of Stomatology, 2016, 25(5): 553-559.

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