粪肠球菌脂磷壁酸对人牙周膜成纤维细胞表达Toll样受体2及白细胞介素-1β的影响

目的 探讨体外培养的人牙周膜成纤维细胞(HPDLCs)在粪肠球菌脂磷壁酸(LTA)刺激下表达Toll样受体2(TLR2)及白细胞介素-1β (IL-1β)的情况.方法 体外分离培养HPDLCs,采用流式细胞术检测0.1、1、10μg·mL-1粪肠球菌LTA刺激24h后HPDLCs表面TLR2表达的改变,酶联免疫吸附法检测12、24、48h后IL-1β的分泌情况,并用相同质量浓度的大肠杆菌内毒素作为对照;用TLR2中和抗体预处理HPDLCs 1 h,观察1μg·mL-1 LTA刺激24h后其IL-1β的分泌量.结果 LTA可致HPDLCs表面TLR2表达增加(P＜0.05);与对照组相比,粪肠球菌LTA可致HPDLCs分泌IL-1β增加(P＜0.05),刺激12h后可检测到IL-1β,48 h内呈上升趋势.TLR2中和抗体对粪肠球菌LTA诱导HPDLCs分泌IL-1β无明显封闭作用.结论 粪肠球菌LTA可引起HPDLCs表面TLR2表达及IL-1β分泌增加.     

粪肠球菌脂磷壁酸对巨噬细胞自噬的影响

目的通过研究粪肠球菌脂磷壁酸（LTA）对巨噬细胞自噬功能的影响,为后续进一步深入探讨顽固性根尖周炎的发病机制提供实验依据。 方法体外培养小鼠巨噬细胞系RAW 264.7,细胞计数试剂盒（CCK-8）检测LTA的细胞毒性作用;蛋白免疫印迹法（Western blot）检测LTA作用下巨噬细胞自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值、Beclin1的表达水平;实时荧光定量聚合酶链反应（PCR）检测自噬相关基因LC3、Beclin1 mRNA表达水平的变化;构建稳定表达自噬双荧光慢病毒载体HBLV-mRFP-GFP-LC3-PURO的巨噬细胞系,激光共聚焦显微镜观察自噬情况;SPSS 20.0统计软件进行数据分析。 结果CCK-8结果表明,选用20 μg/mL LTA及其以下浓度处理巨噬细胞24 h均未见明显毒性作用（t = 2.102,P = 0.1106）;Western blot结果表明,LTA刺激巨噬细胞后：LC3-Ⅱ/LC3-Ⅰ比值表达量（0.42 ± 0.04）与对照组（0.04 ± 0.02）相比显著提高,差异有统计学意义（F = 14.25,P<0.001）,Beclin1蛋白表达量（0.56 ± 0.11）与对照组（0.28 ± 0.08）相比呈上升趋势（F = 3.459,P = 0.0258）,均存在剂量依赖效应;实时荧光定量PCR结果表明,LTA刺激巨噬细胞后,LC3基因表达水平（5.94 ± 0.85）与对照组（1.03 ± 0.28）相比显著提高（F = 12.93,P<0.001）;Beclin1基因表达水平（2.22 ± 0.21）与对照组（1.02 ± 0.28）相比呈上升趋势（F = 6.036,P<0.001）,存在剂量依赖效应;成功构建稳定表达HBLV-mRFP-GFP-LC3-PURO的巨噬细胞系,激光共聚焦显微镜下观察显示,LTA作用组自噬体形成量（58 ± 6）与对照组（18 ± 8）相比差异有统计学意义（F = 12.36,P = 0.0021）。 结论粪肠球菌LTA可诱导巨噬细胞产生自噬,且存在剂量依赖效应。     

粪肠球菌和变异链球菌脂磷壁酸的生物学活性

粪肠球菌是难治性根尖周炎根管内的主要致病菌。变异链球菌是引起龋齿的主要致病菌。粪肠球菌和变异链球菌的毒力因子脂磷壁酸（LTA）皆可促进细胞分泌炎症因子,引发炎症反应,促进根尖周牙槽骨吸收;有助于细菌黏附于牙体表面和生物膜的形成。粪肠球菌和变异链球菌LTA在细菌致病性方面起着重要的作用,但其机制尚不清楚。因此,研究LTA的致病机制及分子结构,旨在实现阻断其致病通路、降低其致病性。     

TRAF6在粪肠球菌感染人成骨样细胞炎症反应中的作用

目的 探讨肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)在粪肠球菌引发的人成骨样细胞MG63炎症反应中的作用.方法 采用siRNA瞬时干扰沉默MG63细胞的TRAF6基因,用粪肠球菌灭活全菌及其脂磷壁酸(lipoteichoic acid,LTA)刺激MG63细胞不同时间,定量PCR检测细胞中Toll样受体2(toll-like receptor 2,TLR2)和TRAF6的表达量;ELISA法检测MG63细胞产生的致炎因子白细胞介素1β(interleukin 1β,IL-1β)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达量.结果 粪肠球菌及其LTA感染MG63细胞,TLR2受体和TRAF6的基因水平都有不同程度的上升(P<0.05).IL-1β和TNF-α的表达量都显著增高(P<0.05).用siRNA抑制MG63细胞TRAF6表达后,促炎因子IL-1β和TNF-α的表达量显著下降(P<0.05).结论 MG63细胞主要通过TLR2受体来识别粪肠球菌及其毒性成分.粪肠球菌引起的根尖周感染中,其主要毒力因子是其细胞壁上的LTA.     

内毒素对人牙周膜细胞Toll样受体2和Toll样受体4表达的影响

目的:观察内毒素(LPS)刺激后人牙周膜细胞Toll样受体2和Toll样受体4的表达,探讨牙周膜细胞作为免疫活性细胞在牙周炎局部免疫反应中的作用。方法:组织块法原代培养人牙周膜细胞,经来源鉴定后,取生长良好的第4代细胞用LPS进行刺激,分别于刺激0、4、8、12、24 h,运用免疫细胞化学染色法检测TLR2和TLR4在牙周膜细胞中的表达;利用多功能真彩色细胞分析管理系统进行图像分析,对TLR2和TLR4进行免疫组化评分(IHS)。结果:无LPS刺激的对照组(刺激O h)TLR2和TLR4的表达均为弱阳性,加入LPS后,染色增强,且随着刺激时间的延长而增加(P<0.05),LPS刺激后各时间点TLP2和TLR4的IHS分值与对照组相比差异均有统计学意义(P<0.05)。结论:内毒素的刺激可以使牙周膜细胞TLR2和TLR4表达增多,提示牙周膜细胞参与牙周局部免疫反应。     

白细胞介素-1β对人牙周膜成纤维细胞中MCP-1表达影响的研究

目的:探讨白细胞介素-1β(IL-1β)对人牙周膜成纤维细胞(hPDLFs)中单核细胞趋化蛋白-1(MCP-1)表达水平的影响。方法:体外分离培养的人牙周膜成纤维细胞随机分为实验组和对照组,实验组细胞以不同浓度IL-1β(1、5、10、25 ng/mL)作用24 h;对照组细胞则不加IL-1β作用。分别采用半定量逆转录聚合酶链反应(RT-PCR)检测人牙周膜成纤维细胞中MCP-1 mRNA的表达;采用免疫荧光技术观察MCP-1的表达情况;采用Western blot方法检测其蛋白表达变化。结果:对照组hPDLFs中可见微弱的MCP-1表达;实验组hPDLFs经IL-1β刺激后,MCP-1在mRNA和蛋白水平表达都增强,这种调节作用在10 ng/mL IL-1β的作用浓度时最明显(P<0.05)。结论:在IL-1β介导环境下,hPDLFs中MCP-1的表达增高,而MCP-1表达增高可能是引起外周血单核细胞向局部炎症牙周组织募集的机制之一。     

牙周状况、血清白细胞介素-1β水平与妊娠关系初探

目的探讨牙周状况、血清白细胞介素- 1β（IL- 1β）水平与妊娠间的关系。方法以40例先兆早产孕妇（TPL组）和40例正常孕妇（Non- TPL组）为研究对象，检查全口牙齿的牙周状况，记录菌斑指数（PLI）、探诊深度、临床附着丧失和出血指数（BI），并计算牙周炎位点率。酶联免疫吸附试验检测血清IL- 1β水平。对各项检查指标进行统计分析。结果①TPL组40例孕妇中26例足月产（TPL- TB小组），14例早产（TPL- PB小组）；Non- TPL组40例孕妇皆足月产。TPL组和Non- TPL组、TPL- TB小组和TPL- PB小组的分娩孕周、新生儿出生体重、PLI、牙周炎位点率和血清IL- 1β水平间的差异均有统计学意义（P<0.05）。②分娩孕周和牙周炎位点率、BI、IL- 1β水平呈负相关（P<0.05），IL- 1β水平与牙周炎位点率和BI呈正相关（P<0.05）。结论牙周感染可能是早产的原因之一。     

乳铁蛋白下调脂多糖激发的人牙周膜细胞Toll样受体4表达的研究

目的 观察乳铁蛋白（LF）对经脂多糖（LPS）刺激的人牙周膜细胞（hPDLCs）表达Toll样受体4（TLR4）的影响。方法 采用组织块酶消化法培养hPDLCs,鉴定后取第4代细胞,分成空白对照组、LPS组、LPS+LF组。空白对照组不加任何刺激,LPS组加入0.1 μg?mL-1 LPS;LPS+LF组在加入0.1 μg?mL-1 LPS 2 h后,加入10 μg?mL-1 LF。以加入LF时开始计算时间,4 h后采用实时定量聚合酶链反应（RT-PCR）法检测hPDLCs中TLR4 mRNA的表达,24 h后采用细胞免疫荧光染色法观察TLR4蛋白的表达。结果 RT-PCR检测显示：LPS+LF组TLR4 mRNA表达较LPS组明显降低（P<0.05）,与空白对照组无明显差异（P>0.05）。细胞免疫荧光染色法显示：LPS+LF组TLR4蛋白的表达强度较LPS组减弱（P<0.05）,与空白对照组无明显差别（P>0.05）。结论 LF可以下调LPS激发的hPDLCs中TLR4的表达,在牙周炎症TLR4信号通路的调控过程中有一定的作用。     

脂多糖和转化生长因子-β1对牙髓细胞Toll样受体4的表达及信号通路的影响

目的研究牙髓炎症过程中,在促炎因子脂多糖（LPS）和抑炎因子转化生长因子-β1（TGF-β1）同时存在的情况下,牙髓细胞表面Toll样受体4（TLR4）的表达水平及相关信号分子的变化情况。方法LPS、TGF-β1作用于体外培养的牙髓细胞,用流式细胞术检测牙髓细胞表面TLR4的表达;实时荧光定量聚合酶链反应（real-time PCR）、Westernblot方法检测相关信号分子的表达水平,包括进化保守的Toll信号中介分子（ECSIT）和核转录因子-κB（NF-κB）;酶联免疫吸附试验（ELISA）检测前炎症因子白细胞介素-6（IL-6）的分泌水平;然后进一步通过real-time PCR法检测临床炎症牙髓组织中相应指标的变化。结果体外培养的牙髓细胞在LPS、TGF-β1共同作用下,细胞表面TLR4的表达水平没有明显变化,但是IL-6分泌增加,ECSIT表达增加,NF-κB入核增加。临床标本的real-time PCR结果表明：炎症状态下的牙髓组织中TGF-β1 mRNA表达增加,TLR4 mRNA表达没有明显变化,ECSIT及IL-6 mRNA表达增加。结论牙髓炎症发展过程中,虽然牙髓组织中TGF-β1表达增加,抑制细胞表面TLR4的表达,但TLR4的信号通路仍然被活化,主要机制可能是LPS引起信号分子ECSIT的活化,从而抑制TGF-β1信号通路的活化。     

LncRNA MAFG‐AS1 regulates human periodontal ligament stem cell proliferation and Toll‐like receptor 4 expression

LncRNA MAFG‐AS1 is predicted to interact with miR‐146a, which can target Toll‐like receptor 4 (TLR4), a key player in periodontitis. This study aimed to investigate the roles of MAFG‐AS1 in periodontitis. It was observed that MAFG‐AS1 was downregulated in the human periodontal ligament stem cells (PDLSCs) derived from periodontitis‐affected teeth. Dual‐luciferase assay revealed that co‐transfection of MAFG‐AS1 expression vector and miR‐146a mimic showed significantly lower relative luciferase activity comparing to co‐transfection of MAFG‐AS1 expression vector and negative control (NC) miRNA. However, MAFG‐AS1 and miR‐146a failed to affect each other. Interestingly, MAFG‐AS1 overexpression led to the upregulated TLR4. In addition, MAFG‐AS1 overexpression also led to the inhibited proliferation of PDLSCs. Therefore, MAFG‐AS1 may regulate the proliferation of PDLSCs and the expression of TLR4 to participate in periodontitis.     

In vivo expression of Toll‐like receptor 2, Toll‐like receptor 4, CSF2 and LY64 in Chinese chronic periodontitis patients

Oral Diseases (2010) 16 , 343–350 Objective: Toll‐like receptors (TLRs) are the essential components in the innate and adaptive immune systems. Colony stimulating factor 2 (CSF2) is a cytokine that may prevent endotoxin tolerance, and LY64 has the ability to interfere with the recognition of bacteria via TLR4. The aim of this study was to explore the in vivo expressions of TLR2, TLR4, CSF2 and LY64 in Chinese chronic periodontitis patients. Methods: Gingival biopsies were collected from 24 chronic periodontitis patients and 19 healthy controls. The gene expression profiles of TLR2, TLR4, CSF2 and LY64 were investigated by real‐time polymerase chain reaction, and the protein expressions of TLR2 and TLR4 were detected by immunohistochemistry. In addition, the levels of CSF2 in gingival crevicular fluid (GCF) were determined by ELISA. Results: The higher mRNA expressions of TLR2, TLR4 and CSF2, and the lower mRNA expression of LY64 were detected in chronic periodontitis patients. And the increased protein expressions of TLR2 and TLR4 were confirmed by immunohistochemistry. In addition, the increase of total amount of CSF2 in GCF was observed in chronic periodontitis patients. Conclusions: Our results suggest that TLR2 and TLR4 may play a role in periodontal pathogenesis. In addition, CSF2 and LY64 may contribute to the regulation of inflammatory response and maintaining periodontal homeostasis.     

脂多糖和白细胞介素-1β对人牙周膜细胞中诱导型一氧化氮合酶表达的影响

目的 观察脂多糖(LPS)和白细胞介素-1β(IL-1β)对人牙周膜细胞(hPDLCs)表达诱导型一氧化氮合酶(iGNOS)和一氧化氮(NO)的影响.方法 应用LPS和IL-1β刺激hPDLCs后,通过实时定量PCR检测iNOS基因的表达情况,收集细胞上清液,酶联免疫吸附试验(ELISA)测定诱导后细胞中iNOS的含量...     

Mitogen-activated protein kinases mediate interleukin-1beta-induced receptor activator of nuclear factor-kappaB ligand expression in human periodontal ligament cells

BACKGROUND AND OBJECTIVE: Interleukin-1beta-stimulated receptor activator of nuclear factor-kappaB ligand (RANKL) expression in human periodontal ligament cells is partially mediated by endogenous prostaglandin E2, whereas mitogen-activated protein kinases (MAPKs) are implicated in regulating various interleukin-1-responsive genes. We investigated herein the involvement of MAPKs in interleukin-1beta-stimulated RANKL expression in human periodontal ligament cells. MATERIAL AND METHODS: Human periodontal ligament cells were pretreated separately with specific inhibitors of MAPKs, including extracellular signal-regulated kinase, p38 MAPK and c-Jun N-terminal kinase, and subsequently treated with interleukin-1beta. Following each treatment, the phosphorylation of each MAPK, the expression of RANKL, and the production of prostaglandin E2 were determined. RANKL activity was evaluated using an assay to determine the survival of prefusion osteoclasts. RESULTS: Interleukin-1beta induced RANKL expression at the mRNA and protein levels, as well as RANKL activity in human periodontal ligament cells. Interleukin-1beta also activated extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Pretreatment with each MAPK inhibitor partially, but significantly, suppressed interleukin-1beta-induced RANKL expression and its activity, as well as prostaglandin E2 production. CONCLUSION: In human periodontal ligament cells, three types of MAPK inhibitor may abrogate RANKL expression and activity induced by interleukin-1beta, directly or indirectly through partial suppression of prostaglandin E2 synthesis. In addition, extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase signals may co-operatively mediate interleukin-1beta-stimulated RANKL expression and its activity in those cells.     

Down-regulation of interleukin-1alpha-induced matrix metalloproteinase-13 expression via EP1 receptors by prostaglandin E2 in human periodontal ligament cells

In the present study, we investigated the effect of prostaglandin (PG) E2 on matrix metalloproteinase (MMP)-13 production in human periodontal ligament cells stimulated with interleukin (IL)-1alpha. IL-1alpha enhanced both MMP-13 and PGE2 production. Indomethacin, a nonselective cyclooxygenase inhibitor, and NS-398, a specific cyclooxygenase-2 (COX-2) inhibitor, significantly enhanced IL-1alpha-induced MMP-13 production in periodontal ligament cells, although both the agents completely inhibited IL-1alpha-induced PGE2 production. Exogenous PGE2 reduced IL-1alpha-induced MMP-13 mRNA and protein production in a dose-dependent manner. 17-phenyl-omega-trinor PGE2, a selective EP1 receptor agonist, mimicked the inhibitory effect of PGE2 on IL-1alpha-induced MMP-13 mRNA and protein production. On the basis of these data, we suggest that COX-2-dependent PGE2 down-regulates IL-1alpha-elicited MMP-13 production via EP1 receptors in human periodontal ligament cells. PGE2 may be involved in the regulation of destruction of extracellular matrix components in periodontal lesions.