LPS对人牙周膜细胞Toll样受体4及下游炎症因子表达的影响

目的：观察革兰氏阴性菌外膜成分脂多糖（LPS）对牙周膜细胞（PDLCs）Toll样受体4（TLR4）及下游炎症因子表达的影响。方法：用浓度为10μg／mL的LPS对牙周膜细胞进行刺激,Western Blot检测牙周膜细胞上TLR4受体表达,同时ELISA检测培养基上清中炎症因子TNF－α、IL－1β的含量。结果：加入LPS后,牙周膜细胞上TLR4蛋白表达增强,炎症因子TNF－α、IL－1β含量增多,且随LPS的刺激时间增加而呈上升趋势。结论：LPS促进PDLCs的TLR4表达,并导致炎症因子分泌增加,TLR4信号通路在牙周炎发病过程中具有重要作用。     

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

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、24h,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Oh)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     

大肠杆菌脂多糖影响Toll样受体4在人牙髓细胞的表达

目的:探讨大肠杆菌脂多糖(lippolysacchaide,LPS)对体外培养的人牙髓细胞(human dental pulp cells,HDPCs)Toll样受体4(Toll-likereceptor4,TLR4)表达的影响及TLR4在LPS对牙髓细胞激活中的作用。方法:以大肠杆菌LPS刺激体外培养的人牙髓细胞,运用实时荧光定量RT-PCR和免疫荧光技术分别检测牙髓细胞TLR4mRNA和蛋白的表达。利用抗体阻断和ELISA方法观察TLR4在LPS激活牙髓细胞释放IL-1β中的作用。结果:正常牙髓细胞不表达TLR4,1×10-4g/L大肠杆菌LPS作用HDPCs6、12、24h后均可见TLR4在胞膜/胞质的表达,胞核并不表达TLR4。FQRT-PCR结果表明LPS能明显上调牙髓细胞TLR4mRNA的表达(P<0.001),并具有LPS浓度依赖性。抗体阻断和ELISA结果证实LPS能激活牙髓细胞释放IL-1β(P<0.01),抗TLR4单抗能明显抑制LPS对HDPCs的激活(P<0.05)。结论:正常人牙髓细胞不表达TLR4,大肠杆菌LPS能诱导牙髓细胞表达TLR4mRNA和蛋白。TLR4介导了LPS对牙髓细胞的活化,在LPS对牙髓细胞激活效应中具有重要作用。     

Toll样受体2和4在脂多糖诱导人牙周膜成纤维细胞表达细胞核因子-κB受体活化因子配基中的作用

目的 观察在脂多糖（LPS）刺激下,人牙周膜成纤维细胞（HPDLFs）中Toll样受体2（TLR2）和Toll样受体4（TLR4）表达水平的抑制对其表达细胞核因子-κB受体活化因子配基（RANKL）的影响。方法 选用100 ng·mL-1、1 μg·mL-1、10 μg·mL-1大肠杆菌LPS分别刺激HPDLFs,刺激6、12、24、48 h后,采用酶联免疫吸附试验（ELISA）检测HPDLFs表达RANKL的水平。分别运用不同滴度的anti-TLR2+anti-TLR4、anti-TLR2、anti-TLR4抗体预处理HPDLFs,观察
1 μg·mL-1 LPS刺激下,其RANKL表达水平的变化。结果 LPS刺激HPDLFs 6 h后,即可检测到RANKL的表达,24 h达到顶峰,然后逐渐下降;各LPS质量浓度组的规律基本一致。分别用anti-TLR2+anti-TLR4、anti-TLR2、anti-TLR4抗体预处理HPDLFs,在1 μg·mL-1 LPS刺激下,其产生RANKL的水平明显下降（P<0.05）;3组中,RANKL的表达水平有明显差异（P<0.05）,其中anti-TLR2+anti-TLR4抗体处理组RANKL表达量最少,anti-TLR4抗体处理组次之,anti-TLR2
抗体处理组RANKL的表达量最高。结论TLR2、TLR4均参与了LPS诱导HPDLFs表达RANKL的过程;与anti-TLR2抗体相比,anti-TLR4抗体能更有效地抑制LPS刺激后HPDLFs表达RANKL的能力。     

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。     

Toll样受体4对牙周膜细胞增殖的影响

目的：了解Toll样受体4对人牙周膜细胞增殖的影响。方法：以改良组织块法体外获得人牙周膜细胞,取生长良好的第3代细胞随机分为3组：空白对照组、NC对照组（NC－shRNA转染组）和TLR4－shRNA转染组,并于转染48 h 后用倒置荧光显微镜观察转染效果、用荧光实时定量 PCR检测各组细胞中TLR4mRNA的表达水平;最后将3组细胞分别接种于96孔细胞培养板进行培养,分别于培养24、48、72 h用MTT法检测各组细胞的增殖能力。结果：TLR4－shRNA可明显降低人牙周膜细胞的TLR4mRNA表达水平（P＜0．05）;MTT检测结果显示,TLR4－shRNA转染组在常规培养48、72 h的OD值均明显低于空白对照组和NC对照组（P＜0．05）,而NC对照组的OD值在72 h时明显低于空白对照组（P＜0．05）。结论：TLR4对牙周膜细胞的增殖有一定调控作用,转染慢病毒后该作用增强。     

茶多酚对内毒素作用下人牙周膜细胞分泌和表达Toll样受体4的影响

目的:观察茶多酚(tea polyphenol,TP)对内毒素(lipopolysaccharide,LPS)作用下人牙周膜细胞(periodontal ligament cells, PDLCs)分泌和表达Toll样受体4(toll-like receptor 4,TLR4)的影响。方法:体外分离及培养PDLCs,实验组分别为LPS和不同质量浓度的TP的不同组合,对照组为仅含1% FBS的DMEM培养液。培养24 h、48 h和72 h后,通过酶联免疫吸附测定法检测TLR4的分泌量,荧光实时定量PCR法检测TLR4的表达。结果:100 mg/L LPS组PDLCs TLR4分泌量显著高于其它各组(P<0.05),加入TP进行干预后实验组与对照组TLR4的分泌量和表达量无显著差异(P>0.05)。结论:TP对LPS作用下PDLCs TLR4的分泌和表达有一定的抑制作用。     

Toll样受体4对人牙周膜成纤维细胞表达p-IRAK1和p-IкB-α的影响

目的:观察Toll样受体4(toll-like receptors 4,TLR4)对人牙周膜成纤维细胞(human periodontal ligamentcells,HPDLCs)在内毒素脂多糖(lipopolysaccharides,LPS)刺激下表达磷酸化IRAK1(phospho-IRAK1,p-IRAK1)和磷酸化IкB-α(phospho-IкB-α,p-IкB-α)的影响。方法:采用Western印迹和图像分析技术,检测HPDLCs受大肠杆菌LPS(1μg/ml)刺激2.5、5、10和15min后表达p-IRAK1和p-IkB-α的水平;同时检测1∶100滴度的抗TLR4单克隆抗体对HPDLCs在1μg/mlLPS刺激下表达p-IRAK1和p-IкB-α的影响。采用SPSS10.0软件包对结果进行单因素方差分析。结果:LPS刺激HPDLCs 5min后,HPDLCs表达p-IRAK1和p-IкB-α的水平最高,条带灰度与3-磷酸甘油醛脱氢酶(GAPDH)条带的比值分别由0.054、0.19增加到0.785、0.809(P<0.05)。抗TLR4单克隆抗体预处理的HPDLCs在LPS刺激下表达p-IRAK1和p-IкB-α的水平均降低,条带灰度与GAPDH条带的比值分别由0.82、0.874降低到0.099、0.201(P<0.05)。结论:TLR4参与了HPDLCs受LPS刺激后的信号传导过程,可能介导了牙周病的发生和发展。     

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样受体与牙周病

Toll样受体是新近发现的先天性免疫系统中的细胞跨膜受体及病原模式识别受体,它通过活化一系列与免疫反应相关的基因而发挥作用,在进化中表现高度的保守性。近年来牙周病与Toll样受体的相关研究日益增多,本文就Toll样受体的研究概况及其与牙周病的关系作一综述。     

TLR4对牙周膜细胞分泌IL-8影响的实验研究

目的:观察TLR4在牙周膜细胞分泌IL-8过程中的作用。方法:采用RT-PCR和ELISA的方法检测IL-8mRNA和蛋白在牙周膜细胞中的表达,并观察不同剂量LPS对其表达量的影响以及抗TLR4抗体作用下IL-8mRNA和蛋白表达量的变化。结果:牙周膜细胞中存在IL-8mRNA表达,分泌少量IL-8蛋白;经LPS刺激后IL-8表达量上调,抗TLR4抗体能够拮抗这一作用。结论:牙周膜细胞表达IL-8mRNA及蛋白,在受到LPS刺激后,IL-8mRNA和蛋白的表达量增加,抗TLR4受体能够拮抗这一作用,提示TLR4参与了牙周膜细胞受到LPS刺激后分泌IL-8的过程。     

TLR4 and TLR9 are induced in oral lichen planus

J Oral Pathol Med (2012) 41 : 741–747 Background: The role of Toll‐like receptors (TLRs) has been elucidated in many human infectious, autoimmune and neoplastic diseases. Previously, TLR2 and TLR4 expression in oral lichen planus (OLP) was described. The aim of our study was to examine expression patterns of TLR4 and TLR9 in normal oral mucosa and OLP and describe the effect of topical tacrolimus treatment on the expression of TLR4 and TLR9 in OLP. Methods: Toll‐like receptor 4 and TLR9 expression was analysed by immunohistochemistry in five samples of normal oral mucosa and 50 samples of OLP (31 representing clinically white and 19 clinically erythematous/erosive lesions). We evaluated also the effect of topical tacrolimus on TLR4 and TLR9 expression in a patient with OLP. Results: Toll‐like receptor 4 and TLR9 expression was increased in OLP epithelium compared with normal epithelium (P < 0.001); no significant difference between the two clinical types of OLP was observed. TLR9 expression was strongest in the superficial layer of the epithelium (P < 0.001), while the expression of TLR4 was strongest in the basal layer (P < 0.001). Treatment of OLP lesions with topical tacrolimus resulted in clinical improvement but had no effect on TLR expression levels. Conclusions: Toll‐like receptor 4 and TLR9 are induced in OLP; our finding confirms the results of a previous study. TLR4 and TLR9 may play a part in the pathogenesis of OLP. Further studies are needed to dissect the definitive role of TLRs in OLP pathogenesis and progression and to determine the effect of tacrolimus on the function of TLRs.     

Expression of transient receptor potential vanilloid receptor 1 and toll-like receptor 4 in aggressive periodontitis and in chronic periodontitis

Öztürk A, Y?ld?z L. Expression of transient receptor potential vanilloid receptor 1 and toll‐like receptor‐4 in aggressive periodontitis and in chronic periodontitis. J Periodont Res 2011; 46: 475–482. © 2011 John Wiley & Sons A/S Background and Objective: The objective of the present study was to evaluate the expression and the distribution of the transient receptor potential vanilloid receptor 1 (TRPV1) and of toll‐like receptor 4 (TLR4) in tissue samples from patients with periodontal disease (aggressive periodontitis and chronic periodontitis) and from healthy controls. Material and Methods: Ten tissue samples from each disease group (aggressive periodontitis and chronic periodontitis) and from healthy subjects were obtained during routine oral surgical procedures. Subgingival specimens were collected from sites with advanced loss of support (probing depth > 5 mm) and specimens from the corresponding healthy controls were obtained during tooth extraction for orthodontic reasons or following surgical extraction of an impacted third molar. The distribution of TRPV1 and TLR4 receptors in human gingival tissue was studied by immunohistochemistry. Results: Both TLR4 and TRPV1 were detected in gingival tissues from healthy subjects, and from patients with chronic periodontitis and aggressive periodontitis, particularly in gingival keratinocytes, fibroblasts, inflammatory cells and the endothelial lining of capillaries in connective tissues. Histologic examination of the samples from healthy controls disclosed that clinically healthy gingiva does not correspond to histologically healthy gingiva. Subsequently, these samples were redesignated as gingivitis samples. TRPV1 was down‐regulated in all cell types in samples obtained from patients with chronic periodontitis compared to samples obtained from patients with gingivitis, whereas TLR4 was down‐regulated only in the epithelium and in gingival fibroblasts. In contrast, the levels of these markers in patients with aggressive periodontitis were similar to those in healthy patients. Conclusion: Local expression of TRPV1 and TLR4 in gingival tissues may contribute to both physiological and pathological processes in the periodontium. Our data suggest that TRPV1 and TLR4 may play a role specifically in the pathophysiology of chronic periodontitis.     

Combined effects of hydrogen sulfide and lipopolysaccharide on osteoclast differentiation in rats

Background: Lipopolysaccharide (LPS) stimulates osteoclast differentiation through toll‐like receptors (TLRs) 2 and 4, and hydrogen sulfide (H₂S) induces osteoclast differentiation. If H₂S activates TLRs, H₂S may enhance the effects of LPS on osteoclast differentiation. The purpose of the present study is to examine the combined effects of sodium hydrogen sulfide (NaHS, an H₂S donor drug) and LPS on osteoclast differentiation and TLR expression in rat periodontal tissue. Methods: Twenty‐eight male Wistar rats (8 weeks old) were divided into four groups (n = 7 per group): a control (no treatment) group and three experimental groups (NaHS group, LPS group, and a combination [NaHS + LPS] group). At 1 day after topical application of NaHS and/or Porphyromonas gingivalis LPS into the gingival sulcus of first molars, the number of tartrate‐resistant acid phosphate (TRAP)‐positive osteoclasts in the periodontal tissue was counted. Expression of TLR2 and TLR4 mRNAs and proteins in the gingival was also assessed. Results: The number of TRAP‐positive osteoclasts was significantly higher in the combination group than in any other group (P <0.01). The combination group had 11.0‐fold higher TLR4 mRNA levels than the control group. TLR4 protein levels were also higher in the combination group than in the NaHS or LPS group. However, the TLR2 mRNA and protein levels were not significantly different in the combination group and the LPS group. Conclusion: In rat periodontal tissue, NaHS and LPS had an additive effect on osteoclast differentiation through activation of the TLR4 pathway but not the TLR2 pathway.     

Toll样受体4在大鼠炎症牙髓表达的免疫组化研究

目的研究内毒素(LPS)诱导大鼠牙髓炎模型中Toll样受体4(TLR4)的表达特点,以探讨TLR4在牙髓炎症中的可能作用,推测它在牙髓炎发生发展中的意义。方法通过对LPS诱导的大鼠磨牙牙髓炎组织切片,同时采用免疫组化方法检测大鼠牙髓炎中TLR4的表达情况。结果 1 d组:成牙本质细胞呈中度阳性表达,牙髓成纤维细胞为中度至强阳性表达;3⁷ d组:成牙本质细胞、牙髓成纤维细胞阳性表达减弱;2周组:坏死区扩大呈均质弱阳性表达,成牙本质细胞、牙髓成纤维细胞及内皮细胞减少,呈阳性,修复牙本质呈弱阳性表达;3周组:大部分坏死牙髓组织呈均质弱阳性表达。结论 TLR4在大鼠牙髓炎发生、发展呈动态表达,TLR4可能参与调节牙髓炎发生、发展及修复过程。     

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。     

年龄对小鼠腹腔巨噬细胞TLR2/4表达水平及功能的影响

目的：观察不同年龄组小鼠腹腔巨噬细胞Toll样受体2/4（TLR2/4）表达水平的差别,以及该细胞在脂多糖（LPS）刺激下,炎症因子TNF-α分泌水平的差别。方法：采用real-time PCR和流式细胞技术,检测低龄组和高龄组小鼠腹腔巨噬细胞TLR2/4 mRNA和蛋白表达水平的差异;同时检测1μg/mL大肠杆菌（E.coli）LPS或1mg/L牙龈卟啉单胞菌（P.gingivalis）LPS刺激后,两组细胞TLR2/4表达水平的变化;采用ELISA技术检测炎症因子TNF-α分泌水平的变化。结果：刺激前,两组细胞TLR2/4 mRNA和蛋白表达水平均无明显差别。E.co-li LPS或P.gingivalis LPS刺激后,低龄组细胞TLR2/4mRNA和蛋白表达水平均明显高于高龄组（P〈0.05）,其分泌的TNF-α水平也显著高于高龄组（P〈0.05）。结论：年龄对小鼠腹腔巨噬细胞TLR2/4表达水平没有显著影响,但增龄性变化可能导致TLR2/4功能的减退。