Toll样受体2和4在细胞成骨向分化中的作用

Toll样受体（TLR）4是人类发现的第一个TLR相关蛋白质,主要介导格兰阴性细菌的免疫过程。TLR2具有相对广泛的配体特异性,可识别多种病原体相关分子模式,协助TLR4参与人体内对脂多糖的反应。研究显示, TLR2和TLR4参与多种细胞成骨向分化的调控。譬如TLR2和TLR4即可通过促进主动脉瓣间质细胞的成骨向分化参与主动脉瓣钙化过程,也可通过上调冠状动脉内皮细胞中骨形态发生蛋白2的表达参与冠状动脉粥样硬化,还可参与骨髓间质干细胞的成骨向分化。在牙髓组织中,有TLR4表达的成牙本质细胞样细胞可向成牙本质细胞受损处迁移并形成修复性牙本质;而TLR2和TLR4作为参与牙髓防御反应的成员,在牙髓组织受损时,不仅可调节炎症因子的表达,还参与牙髓细胞成牙本质向分化的调控。由此可见,TLR2和TLR4在细胞成骨向分化中的作用机制值得进一步的探讨。     

Dickkopf-1基因在头颈部鳞状细胞癌中的表达及对细胞系表型变化的影响

目的 通过数据库分析Dickkopf-1(DKK1)基因在头颈部鳞状细胞癌（head and neck squamous cell carcinoma,HNSCC）中的表达和临床意义，DKK1对头颈部鳞癌细胞表型的影响。方法 使用GEPIA分析DKK1在HNSCC中的表达及其与患者生存率间的关系，并通过qRT-PCR对DKK1在HNSCC细胞系中的表达进行验证。使用LinkedOmics数据库分析DKK1表达与HNSCC临床病理特征关系。使用siRNA转染下调HNSCC细胞系CAL-27细胞中DKK1表达并通过流式细胞术、细胞划痕实验和细胞侵袭实验评估DKK1对头颈部鳞癌细胞增殖、迁移和侵袭的影响。结果 DKK1在HNSCC中显著高表达；qRT-PCR体外验证DKK1在HNSCC细胞系中表达均升高，其中在SCC-4、SCC-9、SCC-25、CAL-27中表达显著升高。DKK1表达水平与HNSCC患者T分期和病理分期呈显著正相关，与HNSCC患者总生存率呈显著负相关。下调DKK1表达后，CAL-27细胞增殖、迁移和侵袭能力明显减弱。结论 DKK1与HNSCC的发生发展及预后密切相关，可...     

人乳头瘤病毒相关头颈部鳞状细胞癌侵袭转移的分子机制

人乳头瘤病毒（HPV）是头颈部鳞状细胞癌（HNSCC）的主要致病因素之一,HPV感染与HNSCC的侵袭转移存在密切关系。本文对HPV相关性头颈部鳞状细胞癌基因变异、HPV致癌蛋白E6、E7通过众多复杂细胞元件相互作用的特异性致癌机制进行了阐述,并进一步讨论了HPV阳性HNSCC侵袭转移相关的分子机制,包括非编码RNAs、肿瘤细胞的能量代谢紊乱、肿瘤细胞微环境、肿瘤干细胞、肿瘤血管生成及淋巴管生成的研究进展。     

mTOR调控口腔鳞状细胞癌增殖转移的机制研究

目的：研究Toll样受体4（TOLL-like receptor 4,TLR4）分子高表达与口腔鳞状细胞癌预后的关系,探讨哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）通过TLR4信号通路间接调控口腔鳞状细胞癌增殖转移的潜在机制。方法：采用免疫组织化学法检测50例口腔鳞状细胞癌患者病理切片中TLR4的表达;使用mTOR抑制剂(雷帕霉素)作用于口腔鳞状细胞癌细胞系CAL27后,再用LPS激活TLR4信号通路,通过MTT法检测CAL27细胞的增殖能力,Transwell法检测CAL27细胞的迁移能力, Western 免疫印迹检测其对肿瘤细胞中NF-κB 及MAPK信号通路的影响。采用SPSS 16.0软件包对数据进行统计学分析。结果：TLR4分子高表达与口腔鳞状细胞癌预后差显著相关（P<0.05）;雷帕霉素能够显著抑制TLR4激活后的CAL27细胞的增殖和迁移能力（P<0.01）,显著抑制TLR4下游的NF-κB 及MAPK信号通路（P<0.01）。结论： mTOR通过TLR4信号通路调控口腔鳞状细胞癌的增殖与转移,有望成为治疗口腔鳞状细胞癌的新靶点。     

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。     

TLR9在成牙本质细胞中的表达

目的：研究成牙本质细胞中TLR9、DSPP基因表达特征及信号转导途径。方法：采用RT-PCR检测TLR9在小鼠牙髓组织中及TLR9、DSPP在成牙本质细胞系中的表达。用CpGODN-A和CpGODN-B刺激细胞,在时间梯度0、3、6、9、12、24h,检测TLR9、DSPP基因的表达特征。结果：TLR9mRNA在小鼠牙髓组织中有表达。TLR9、DSPPmRNA在成牙本质细胞中都有显著的表达,在CpGODN刺激下显著上调,在6h处于峰值。结论：TLR9在成牙本质细胞中有表达;TLR9的特异性表达对DSPP基因表达量有影响。     

模式识别受体和病原体相关分子模式及其在牙周病防御中的作用

模式识别受体(PRR)是宿主抵抗病原微生物的感应器,可识别一种或多种病原体相关分子模式(PAMP),可及时向下游通路转导信号,引发先天性免疫反应.PAMP包括脂多糖(LPS)、脂磷壁酸、肽聚糖和甘露糖等.不同的PAMP可被不同的Toll样受体(TLR)识别或组合识别,然后通过一系列蛋白质级联反应激活细胞因子,从而有效地活化天然免疫系统.PAMP作用于TLR后,可促进炎性细胞因子的合成与释放;诱导一氧化氮依赖性杀菌活性和呼吸爆发;介导由细菌脂蛋白引起的人单核细胞系和表达TLR2的上皮细胞系程序性细胞死亡;LPS通过作用于TLR4促进树突细胞成熟,诱导TLR2的合成与表达,进而分泌白细胞介素-6等细胞因子.对TLR的研究将有助于认识慢性牙周病并为其治疗提供新的方法,故本文就牙龈上皮表面的PRR和牙周致病菌表面的PAMP及其在牙周病中的防御作用等研究进展作一综述.     

炎症信号受体TLR4在大鼠正常牙髓和牙周组织中的分布及意义

目的:研究大鼠正常牙髓和牙周组织中炎症信号受体TLR4表达特点,探讨其在健康牙髓及牙周组织天然免疫防御中的可能作用.方法:采用石蜡及冰冻切片和免疫组化技术检测大鼠健康牙髓及牙周组织中TLR4的表达情况.结果:正常牙髓组织中成牙本质细胞、血管内皮细胞及参与先天免疫的巨噬细胞、树突状细胞、中性粒细胞等细胞TLR4免疫反应阳性,牙髓成纤维细胞不表达TLR4.TLR4表达于牙周结缔组织,可见与上皮层临近的固有层结缔组织强阳性染色.结论:TLR4可能参与了牙髓牙周组织的先天免疫反应,在牙髓牙周组织抵御外来病原微生物入侵中可能起重要作用.     

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。     

Toll样受体2和4信号通路在炎症治疗中的作用和意义

脂多糖（LPS）在细菌破坏细胞的过程中起着重要的作用。Toll样受体（TLR）2对LPS的识别是通过与TLR1和TLR6构成异源二聚体来完成的,TLR2识别LPs后介导的细胞内免疫反应遵循髓样分化因子（MyD）88依赖性通路。MyD88的死亡结构域募集下游的白细胞介素-1受体相关激酶1和4,肿瘤坏死因子受体相关因子6和转化生长因子-B1活化激酶等信号分子,促使核因子-KB、激活蛋白1和P38促丝裂原激活蛋白激酶活化,继而导致促炎症细胞因子相关基因转录。MyD88非依赖性通路分别募集和激活下游分子受体相互作用蛋白1或肿瘤坏死因子受体相关因子3,通过核因子-κB、激活蛋白1和干扰素调节因子3,诱导Ⅰ型干扰素的产生。CD14和MyD2是LPS与TLR4结合的关键蛋白,控制CD14或MyD2可阻止LPs和TLR4的结合,将炎症反应阻断在信号转导的上游。TLR2和TLR4对LPS的识别是引发炎症反应的关键,限制细胞对TLR2和TLR4的表达是进行炎症控制最直接有效的方法。调控TLR2和TLR4信号通路,有望给予牙周炎、炎症性肠炎、心血管疾病及和自身免疫性疾病等更有效和更安全的临床治疗。     

Toll‐like receptors 2 and 5 in human gingival epithelial cells co‐operate with T‐cell cytokine interleukin‐17

Background/aim: Periodontitis begins as the result of perturbation of the gingival epithelial cells caused by subgingival bacteria interacting with the epithelial cells via pattern recognition receptors. Toll‐like receptors (TLRs) have been shown to play an important role in the recognition of periodontal pathogens so we have studied the interaction of TLR ligands with TLR2 and TLR5 for cytokine production in the cultures of gingival epithelial cells. Methods: Immunohistochemistry was used for the localization of TLR2 and TLR5 in tissue specimens. Enzyme‐linked immunosorbent assays were performed to detect the levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), released from gingival epithelial cell cultures following stimulation with TLR ligand alone or in combination with IL‐17. Results: Both TLR2 and TLR5 were increased in periodontitis (2128 ± 159 vs. 449 ± 59 and 2456 ± 297 vs. 679 ± 103, respectively, P < 0.001) including gingival epithelial cells that stained strongly. Cultured gingival epithelial cells stimulated with their respective ligands (HKLM, a TLR2 ligand that is also found in Porphyromonas gingivalis, and flagellin, a TLR5 ligand that is also found in Treponema denticola) produced both IL‐1β and TNF‐α. To mimic T‐cell help, IL‐17 was added. This further greatly enhanced TLR ligand‐induced IL‐1β (P < 0.001) and TNF‐α (P < 0.01) production. Conclusions: These findings show how pathogen‐associated molecular patterns, shared by many different periodontopathogenic bacteria, stimulate the resident gingival epithelial cells to inflammatory responses in a TLR‐dependent manner. This stimulation may be particularly strong in periodontitis and when T helper type 17 cells provide T‐cell help in intercellular cooperation.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Analysis of Subgingival Plaque Ability to Stimulate Toll‐Like Receptor 2 and 4

Background: It has been shown that toll‐like receptor (TLR) 2‐ and TLR4‐stimulating abilities of supragingival plaque (SPP) are associated with periodontal conditions. It is hypothesized that SPP might affect the periodontium through its influence on subgingival plaque (SBP). This study investigates relationships between TLR2‐ and TLR4‐stimulating abilities of SBP and periodontal conditions. Methods: One hundred thirteen SBP samples were collected from the deepest pockets in patients with chronic periodontitis. TLR2‐ and TLR4‐stimulating abilities were measured using genetically engineered nuclear factor‐kappa B reporter cells. Numbers of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in each plaque sample were determined by real‐time polymerase chain reaction. Peripheral blood mononuclear cells (PBMCs) were stimulated with SBP samples in presence or absence of TLR4 or TLR2 inhibitor. Production of tumor necrosis factor (TNF)‐α and interleukin (IL)‐8 was analyzed by enzyme‐linked immunosorbent assay. Results: TLR4‐stimulating ability of SBP was associated with plaque index (PI), but not with other clinical parameters at sampling sites. TLR2‐stimulating ability of SBP was associated with none of the parameters. Number of P. gingivalis and A. actinomycetemcomitans in each plaque sample was not associated with TLR2‐ or TLR4‐stimulating ability of SBP. PBMCs stimulated with SBP samples produced TNF‐α and IL‐8, which was inhibited by TLR4 but not by TLR2 inhibitor. Conclusion: TLR4‐ but not TLR2‐stimulating ability of SBP is associated with PI. Enhanced TLR4‐stimulating ability at sites with accumulated plaque may mediate gingival inflammation.     

Toll‐like receptor 5 activation promotes migration and invasion of salivary gland adenocarcinoma

J Oral Pathol Med (2011) 40 : 187–193 Background: Toll‐like receptor (TLR) signaling has been found to be closely associated with tumor development. The aim of this study was to examine whether activation of TLRs promote migration and invasion of salivary gland adenocarcinoma. Materials and Methods: TLR expression in SGT and HSG cells was examined by RT‐PCR. Wound scratch and chemotaxis cell migration assay were performed. Invasiveness was determined by Matrigel invasion assay. Results: All the tested TLRs including TLR1, TLR2, TLR4, and TLR5 and myeloid differentiation factor‐2 (MD‐2) were expressed on SGT and HSG cells. Treatment of flagellin, but not Pam₃CSK₄ and LPS, led to the production of IL‐6 and IL‐8, suggesting TLR5 is functional in both cells. Stimulation by flagellin also accelerated wound closure of SGT and HSG cells in a dose‐dependent manner. In addition, flagellin promoted migration and invasion ability of SGT cells. Blocking of TLR5 using antibody restored the promoting effect of flagellin on migration and invasion of SGT cells. Conclusion: These findings suggest that TLR5 activation by flagellin can promote migration and invasion of salivary gland adenocarcinoma.     

Increased Nucleic Acid Receptor Expression in Chronic Periodontitis

Background: Nucleic acid sensing has emerged as one of the important components of the immune system triggering inflammation. The aim of this study is to determine the expression of bacterial DNA sensors, including Toll‐like receptor 9 (TLR‐9), DNA‐dependent activator of interferon‐regulatory factors (DAI), and absent in melanoma 2 (AIM2) in chronic periodontitis (CP versus healthy) (H) tissues. Methods: Thirty‐five CP and 27 H gingival biopsies were included. Real‐time quantitative polymerase chain reaction was performed to determine mRNA levels of AIM2, DAI, and TLRs (TLR‐1 through TLR‐9). The difference in gene expression for each sensor between CP and H tissues was calculated using analysis of covariance. The Spearman test was used to determine correlations among innate receptors. The expression of TLR‐9, AIM2, and DAI in gingival tissues was further confirmed using immunohistochemistry. Results: The present results reveal statistically significant upregulation of TLR‐9 (P <0.006), DAI (P <0.001), and TLR‐8 (P <0.01) in CP tissues compared to H sites. Although mRNA expression was not changed significantly between groups for other receptors, the present results reveal significant correlations between receptors (P <0.05), suggesting that cooperation between multiple components of the host immune system may influence the overall response. Immunohistochemistry further confirmed expression of TLR‐9, AIM2, and DAI in gingival tissues. Conclusions: This study highlights a possible role for nucleic acid receptors in periodontal inflammation. Future investigations will determine whether cytoplasmic receptors and their ligands can be targeted to improve clinical outcomes in periodontitis.     

Toll-like receptors and their signaling mechanism in innate immunity

In Drosophila the Toll family, a group of transmembrane proteins, plays crucial roles in the host defense against invading pathogens. Mammalian species also conserve this system as the Toll-like receptor (TLR) family, which includes more than 10 members that have been identified so far. Both the Toll and TLR families recognize various kinds of microorganisms through pathogen-associated molecular patterns. Mammalian TLRs are expressed on macrophages and dendritic cells and mediate the signal for cytokine release or upregulation of costimulatory molecules. These activities cooperatively generate host defense mechanisms. Recently, gene targeting experiments, including ours, have contributed much to clarifying not only the function but also the signaling mechanism of TLRs. TLR2 is essential for recognizing lipopeptides and lipoproteins from several microorganisms and also peptidoglycans derived from gram-positive bacteria. TLR4 recognizes lipopolysaccharides and lipoteichoic acids from gram-negative and- positive bacteria, respectively. Furthermore, TLR9 is critical for recognizing bacterial DNAs. Thus, TLRs distinguish various immunostimulatory molecular patterns. Although TLR9 can produce similar biological responses, studies with mutant mice lacking a TLR-associating protein, MyD88, showed that TLR signaling is differentially regulated among TLR family members. Here, we describe recent progress in elucidating the function and signaling mechanisms of the TLR family.     

低氧促进口腔鳞癌细胞TLR3及TLR4表达的相关研究

目的　　检测常氧培养条件下口腔鳞癌细胞HSC3中Toll样受体3、4（Toll like receptor 3,4,TLR3、TLR4）的表达水平,并进一步探讨低氧微环境对其表达可能的影响。方法　常氧条件下,将口腔鳞癌细胞HSC3培养至指数增长期,收集细胞,采用Real-time PCR方法在mRNA水平检测TLR3、4的表达,继之,采用Western blotting在蛋白水平检测其表达。模拟体内低氧微环境,在体外采用1% O2浓度分别刺激细胞0、3、6、12及24 h,采用Real-time PCR方法在mRNA水平检测TLR3、4的表达变化,进一步通过Western blotting在蛋白水平检测其表达改变。结果　在常氧培养条件下, HSC3细胞中可检测到TLR3和TLR4的表达。采用低氧刺激可以显著促进TLR3、TLR4的表达水平,其中低氧刺激24 h后,与对照组相比,实验组TLR3的蛋白表达水平增高至(6.2±0.1)倍,而TLR4在低氧刺激6 h后蛋白表达水平可增高至(5.6±0.1)倍,其结果在统计学上具有显著性差异（P＜0.05）。结论　口腔鳞癌细胞HSC3在常氧培养下可检测到TLR3、TLR4的表达,肿瘤低氧微环境可进一步促进其表达水平。     

Nuclear dot protein 52, an autophagy-associated protein,regulates Toll-like receptor signaling

Toll-like receptors (TLRs) recognize molecular patterns on various microbes and serve as innate immune sensors. After cognate ligand recognition, TLRs activate signaling pathways to induce innate immune defense mechanisms, which eliminate pathogenic microbes, including periodontogenic bacteria, to a certain extent. Recent findings have shown that TLR signaling is linked to induction of autophagy to facilitate direct killing of cytosol-invading bacteria within infected cells. However, whether autophagy has any regulatory effects on TLR signaling remains unclear. Our recent study showed that the signaling molecules Toll/interleukin-1 receptor homology domain-containing adaptor inducing interferon-β and tumor necrosis factor receptor-associated factor 6 are selectively degraded by autophagy after activation of TLR signal transduction. We found that the nuclear dot protein 52 (NDP52), an autophagy-associated protein, is involved in such degradation, negatively regulating TLR signaling. However, interestingly, this activity of NDP52 is strictly restricted by the deubiquitinase A20. Here, we describe an autophagy-associated regulatory function of NDP52 in TLR signaling on the basis of our recent findings.