【作者】 唐路； 周学东； 王倩； 张岚； 王瑶； 李小玉； 黄定明；

【Author】 Lu Tang~2,Xue-dong Zhou~2,Qian Wang~2,Lan Zhang~2,Yao Wang~2,Xiao-yu Li~1,Ding-ming Huang~(*1,2)(1.State Key Laboratory of Oral Diseases,Sichuan University 2.West China school of stomatology,Sichuan University)

【机构】 四川大学华西口腔医学院； 口腔疾病研究国家重点实验室(四川大学)；

【摘要】 目的:探讨人牙周膜成纤维细胞肿瘤坏死因子相关因子6(Tumor necrosis factorreceptor-associated factor 6,TRAF6)在类型识别受体介导的nuclear factor-κB(NF-κB)信号通路调控促炎细胞因子表达的作用。方法:采用逆转录PCR(reverse transcriptionpolymerasechain reaction,RT-PCR)技术、流式细胞术和免疫荧光检测人牙周膜成纤维细胞中TLR2,TLR4,NOD1和NOD2受体表达及其特异性配体刺激后,real-time PCR和western blot检测TRAF6的表达;ELISA法检测人牙周膜成纤维细胞产生的促炎因子(IL-1β,IL-6,and IL-8)表达。小干扰RNA(small interfering RNA,siRNA)技术沉默TRAF6基因,检测TLRs和NODs受体被激活后的人牙周膜成纤维细胞释放促炎因子的表达量。结果人牙周膜成纤维细胞在基因和蛋白水平都稳定表达TLR2,TLR4,NOD1和NOD2受体。人牙周膜细胞TLR2,TLR4,NOD1和NOD2受体激活后,TRAF6和促炎因子(IL-1β,IL-6,and IL-8)的表达上调,并且呈现出时间和浓度依赖性。当TLRs和NODs信号通路被联合激活时,对于TRAF6和促炎因子的表达具有协同增强效应,而当TRAF6的表达受到抑制时,促炎因子(IL-1β,IL-6,and IL-8)的表达量显著下调。结论:人牙周膜成纤维细胞表达的固有免疫识别受体TLR2,TLR4,NOD1和NOD2在识别口腔微生物病原体时发挥了重要的作用。激活TLRs和NODs信号通路会协同性地增强人牙周膜成纤维细胞的炎症反应。人牙周膜成纤维细胞中TRAF6作为一种信号转导蛋白,在TLRs和NODs介导的炎症反应信号通路中起到重要作用。更多还原

【Abstract】 Objectives:Apical periodontitis is an inflammation of dental periapical tissues developed as a response to colonization of microorganisms in root canal system.The periodontal ligament is highly vascularised and cellular connective tissue that anchors the tooth root to the surrounding alveolar bone.Human periodontal ligament fibroblasts (HPDLFs),the dominant cells in the periodontal ligament,encounter bacteria in periapical tissue and produce several pro-inflammatory cytokines.Toll-like receptors(TLRs)and nucleotide-binding oligomerization domain(NOD)are two major forms of innate immune sensors that recognize microbial pathogens and initiate pro-inflammatory signaling. Tumor necrosis factor receptor-associated factor 6(TRAF6)is an adapter protein for TLR-mediated nuclear factor-κB(NF-κB)signaling pathway activation that induces the production of pro-inflammatory cytokines.The aim of this study was to investigate the expression of TLR2,TLR4,NOD1,and NOD2 in HPDLFs.We also investigated the expression of TRAF6 and pro-inflammatory cytokines induced by the activation of TLRs and NODs.Furthermore,by using RNA interference for TRAF6,we analyzed the production of pro-inflammatory cytokines in HPDLFs upon stimulation with TLR and NOD ligands. Methods:The expression of TLR2,TLR4,NOD1,and NOD2 was measured by reverse transcription-polymerase chain reaction(RT-PCR),flow cytometry,and immunostaining. HPDLFs were stimulated with TLR and NOD agonists.Then,the expression of TRAF6 was measured by real-time PCR and western blot.Concentrations of IL-1β,IL-6,and IL-8 in the culture supernatants were determined by enzyme-linked immunosorbent assay(ELISA).Finally,by using small interfering RNA(siRNA)for TRAF6,we analyzed the production of IL-1β,IL-6,and IL-8 in HPDLFs upon stimulation with TLRs and NODs agonists.Results:We found clear mRNA and protein expression of TLR2,TLR4,NOD1, and NOD2 in HPDLFs.The expression levels of TRAF6 and pro-inflammatory cytokines (IL-1β,IL-6,and IL-8)were markedly up-regulated upon the activation of TLRs and NODs.Furthermore,the co-activation of TLRs and NODs had synergistic effect on the production of TRAF6 and pro-inflammatory cytokines.We also found TRAF6 suppression resulted in reduced IL-1β,IL-6,and IL-8 expression upon TLR and NOD agonists challenge.Conclusion:These findings indicated that TLR2,TLR4,NOD1,and NOD2 are functional receptors in HPDLFs during innate immune responses to invading bacteria,and a combination of signaling through TLRs and NODs leads to the synergistic enhancement of inflammatory reactions in HPDLFs.In addition,TLR and NOD signaling involving TRAF6 contribute to inflammatory responses in HPDLFs.更多还原