不同fimA基因型牙龈卟啉单胞菌对人脐静脉内皮细胞产生血管细胞黏附分子1和细胞间黏附分子1的影响

目的 观察不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)产生血管细胞黏附分子1(vascular cell adhesion molecule-1,VCAM-1)和细胞间黏附分子1(intercellular adhesion molecule-1,ICAM-1)的影响,探讨Pg在动脉粥样硬化发生、发展中的可能作用.方法 实验分别以PgATCC33277 (Ⅰ fimA ) 、WCSP115 (Ⅱ fimA)、W83 (Ⅳ fimA)和大肠杆菌脂多糖刺激HUVEC作为T1、T2、T3组(3个实验组)和阳性对照组,未受刺激的HUVEC作为阴性对照组;标准条件下厌氧培养上述3型Pg,将其以及大肠杆菌脂多糖分别与 HUVEC共同孵育2、6、24 h,采用流式细胞术检测HUVEC表面ICAM-1和VCAM-1的蛋白表达量,并通过激光共聚焦显微镜观察ICAM-1和VCAM-1的表达分布情况.结果 Ⅰ、Ⅱ、Ⅳ fimA型Pg刺激HUVEC后,细胞表面ICAM-1表达均增强(P<0.05),2、6、24 h表达量分别为Ⅰ fimA:60.27±5.43、80.81±1.44、85.94±2.56;Ⅱ fimA:86.69±8.81、90.19±0.00、96.18±0.48,Ⅳ fimA:59.66±0.40、85.79±4.86、96.04±2.07.除2 h时ⅠfimA与Ⅳ fimA型Pg刺激的HUVEC表面ICAM-1表达量差异无统计学意义外,其他各时间点Ⅱ、Ⅳ fimA型Pg的刺激作用均强于Ⅰ fimA型Pg(P<0.05).本研究条件下,Ⅰ、Ⅱ、Ⅳ fimA型Pg刺激HUVEC后2、6、24 h表达VCAM-1的水平均较低,各实验组与对照组间相比差异均无统计学意义(P>0.05).激光共聚焦显微镜观察显示,Pg刺激下HUVEC表达ICAM-1和VCAM-1增加,在Ⅱ、Ⅳ fimA型Pg刺激下,HUVEC中ICAM-1和VCAM-1荧光点相对较多且分布范围广.结论 牙周主要致病菌Pg毒力和致病性与其fimA基因型相关,Ⅱ fimA和Ⅳ fimA型Pg 有较强的上调HUVEC表达细胞黏附分子的能力,可能导致血管内皮功能紊乱.

Abstract：

Objective To investigate the effect of Porphyromonas gingivalis(Pg) with different fimA genotypes on vascular cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule-1(ICAM-1) production by human umbilical vein endothelial cells(HUVEC). Methods In the present study, PgATCC33277(type Ⅰ fimA genotype), WCSP 115(type Ⅱ fimA genotype), W83(type Ⅳ fimA genotype), and Escherichia coli-lipopolysaccharide (Ec-LPS) were designed as experimental group 1, 2, 3, and positive control group, respectively, to stimulate HUVEC, and the un-stimulated HUVEC were analyzed as negative control group. The three strains of Pg were cultured anaerobically in standard condition, and then the Pg cells and Ec-LPS were co-cultured with HUVEC for 2, 6, and 24 h, respectively. The amount of ICAM-1 and VCAM-1 produced by HUVEC was detected with flow cytometry(FCM). The expression of ICAM-1 and VCAM-1 by HUVEC were assayed with confocal laser scanning microscope(CLSM). ResultsThe expression of ICAM-1 on the surface of HUVEC were intensified after infected by Pg with Ⅰ, Ⅱ, and Ⅳ fimA genotypes (P<0.05). The amounts of ICAM-1 were 60.27±5.43, 80.81±1.44, and 85.94±2.56 for Pg with type Ⅰ fimA genotype, 86.69±8.81, 90.19±0.00, and 96.18±0.48 for Pg with type Ⅱ fimA genotype, 59.66±0.40, 85.79±4.86, and 96.04±2.07 for Pg with type Ⅳ fimA genotype at 2, 6 and 24 h after infection, respectively. The up-regulation effects caused by Pg with type Ⅱ and Ⅳ fimA genotypes were stronger than those caused by Pg with type Ⅰ fimA genotype at different time points except at 2 h(P<0.05). Under the present experimental condition, infected by Pg with type Ⅰ, Ⅱ and Ⅳ fimA genotypes stimulated low expression of VCAM-1 by HUVEC, it showed no significant differences among all the groups (P>0.05). Expression of ICAM-1 and VCAM-1 in Pg infected HUVEC were confirmed by CLSM. Infection of HUVEC with Pg resulted in more fluorescence staining of ICAM-1 and VCAM-1 compared with that in uninfected HUVEC cultures. Conclusions The virulence and pathogenicity of Pg is associated with its fimA genotypes, Pg with type Ⅱ and Ⅳ fimA genes possess stronger ability to stimulate HUVEC to up-regulate the expression of cell adhesion molecules, which may lead to disorders in vascular endothelial function.     

TP对脂多糖介导人牙周膜成纤维细胞细胞间黏附分子-1表达的影响

目的: 检测人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPDLF)在脂多糖(lipopolysaccharide,LPS)介导下细胞间黏附分子-1﹙intercellular adhesion molecule-1,ICAM-1﹚的表达,探讨TP(tea polyphenols,TP)对其表达的影响。方法: HPDLF采用改良组织块法体外培养,将HPDLF随机分成LPS组、TP100组、TP200组,用酶联免疫吸附试验法(enzyme linked immuosorbent,ELISA)检测不同浓度TP对LPS介导下HPDLF分泌ICAM-1的活性。实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,Real-time PCR)检测ICAM-1 mRNA的表达。结果: 在LPS干预下,不同时间不同浓度的TP影响下均可抑制ICAM-1分泌,其活性降低,与LPS组比较差异有统计学意义(P<0.05);且ICAM-1 mRNA表达水平显著降低,与LPS组比较差异有统计学意义(P<0.05)。结论: TP对ICAM-1的抑制作用明显,100 μg/mL TP对其的抑制作用更显著,提示TP的抗炎机制可能与抑制ICAM-1有关。     

牙龈卟啉单胞菌感染对大鼠血管平滑肌细胞细胞间黏附分子-1表达的影响

目的 观察牙龈卟啉单胞菌ATCC 33277感染对大鼠血管平滑肌细胞（VSMC）细胞间黏附分子-1（ICAM-1）表达的影响。方法 建立体外牙龈卟啉单胞菌感染大鼠VSMC模型,逆转录-聚合酶链反应（RT-PCR）检测ICAM-1基因的表达。结果 牙龈卟啉单胞菌感染VSMC 8、16、24 h后,ICAM-1表达明显增多,与空白对照组比较,差异有统计学意义（P<0.05）。感染16 h达高峰,感染8 h与感染16、24 h比较,差异有统计学意义（P<0.05）。结论 牙龈卟啉单胞菌感染可引起VSMC ICAM-1高表达,这提示牙周致病菌可能参与血管壁的炎症反应,在动脉粥样硬化的发生、发展中有重要的意义。     

肿瘤坏死因子-α、B细胞刺激因子、细胞间黏附分子-1、血管细胞黏附分子-1在原发性舍格伦综合征患者血清中的水平变化

目的探讨原发性舍格伦综合征(primary Sjgeren’s syndrome,p SS)患者血清中肿瘤坏死因子(TNF-α)、B细胞刺激因子(BAFF)、细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)的表达意义,为p SS的临床治疗提供实验依据。方法选择经病理确诊的p SS患者50例,酶联免疫吸附试验(ELISA)检测患者血清中TNF-α、BAFF、ICAM-1、VCAM-1的含量,并利用SPSS软件包对数据进行统计学分析。结果 p SS患者血清中TNF-α、BAFF、ICAM-1、VCAM-1的含量较正常对照组显著升高(P<0.05)。结论细胞因子TNF-α、BAFF、ICAM-1、VCAM-1可能参与了p SS的发生发展过程,其相互作用关系有待进一步研究。     

口腔鳞癌中血管细胞黏附分子-1的表达与巨噬细胞浸润的关系

目的:探讨血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)在口腔鳞癌巨噬细胞浸润、聚集中的作用及意义。方法:应用免疫组化二步法检测口腔鳞癌组织中VCAM-1的表达和巨噬细胞的浸润情况,光镜下进行巨噬细胞计数和VCAM-1表达的分级。结果:口腔鳞癌组织中VCAM-1的表达率(64.58%)和巨噬细胞计数(81.04±12.00)显著高于正常口腔黏膜组织(P<0.05);口腔鳞癌组织中VCAM-1的表达与肿瘤的浸润和转移有关(P<0.01),并与肿瘤组织中巨噬细胞浸润有关(P<0.01)。结论:VCAM-1可能对巨噬细胞在口腔鳞癌中的浸润、聚集起调节作用,参与肿瘤的浸润和转移。     

血管细胞黏附分子-1在口腔鳞状细胞癌中的表达及其意义

目的研究血管细胞黏附分子-1（VCAM-1）在口腔鳞状细胞癌（OSCC）中的表达与定位及其临床意义。方法应用分子原位杂交和免疫组化方法对48例OSCC组织和10例正常口腔黏膜组织中VCAM-1 mRNA和VCAM-1蛋白质的表达和定位进行检测,比较VCAM-1在不同口腔组织中的表达率及其与临床指标的关系。结果VCAM-1蛋白定位于肿瘤细胞胞浆和胞膜,VCAM-1 mRNA定位于肿瘤细胞胞浆。VCAM-1 mRNA和VCAM-1蛋白在OSCC中的表达率均显著高于正常口腔黏膜组织（P＜0.01）,VCAM-1 mRNA表达与VCAM-1蛋白表达呈正相关（P＜0.01）;OSCC中淋巴结转移者VCAM-1蛋白的表达显著高于无淋巴结转移者（P＜0.01）。结论OSCC中VCAM-1基因的高表达可能与肿瘤的浸润和转移有关。     

单核细胞趋化蛋白-1和细胞间黏附分子-1与动脉粥样硬化和牙周病的关系

以动脉粥样硬化为病理基础的冠心病是目前主要的致死性疾病之一,而牙周病与动脉粥样硬化之间存在着一定的相关性。单核细胞趋化蛋白（MCP）-1和细胞间黏附分子（ICAM）-1在动脉粥样硬化和牙周病的发生发展中起着重要的作用。本文就MCP-1和ICAM-1以及二者与动脉粥样硬化、牙周病间的关系作一综述。     

单核细胞趋化蛋白-1和细胞间黏附分子-1与动脉粥样硬化和牙周病的关系

以动脉粥样硬化为病理基础的冠心病是目前主要的致死性疾病之一,而牙周病与动脉粥样硬化之间存在着一定的相关性。单核细胞趋化蛋白(MCP)-1和细胞间黏附分子(ICAM)-1在动脉粥样硬化和牙周病的发生发展中起着重要的作用。本文就MCP-1和ICAM-1以及二者与动脉粥样硬化、牙周病间的关系作一综述。     

口腔扁平苔藓病损区细胞间黏附分子1、干扰素γ表达及意义

目的:探讨细胞间黏附分子1(ICAM-1)和干扰素γ(IFN-γ)在口腔扁平苔藓(OLP)病损形成及发展中的作用和意义.方法:55 例OLP和10 例正常口腔黏膜石蜡包埋组织,采用免疫组化SP法检测ICAM-1和IFN-γ蛋白表达情况,分析两者的相关性及其与OLP临床病理特征的关系.结果: ICAM-1 和IFN-γ在OLP病损中的表达高于正常口腔黏膜中的表达,差异均有统计学意义(P<0.05).OLP上皮组织中ICAM-1的表达与IFN-γ的表达存在正相关关系(P<0.05).IFN-γ和上皮ICAM-1的阳性表达率与OLP基底细胞液化程度存在正相关关系(P<0.05).结论:OLP病损中ICAM-1异常高表达可能与IFN-γ有关,两者协同参与了口腔扁平苔藓病理过程,可能在OLP病损形成中起重要作用.     

不同金属烤瓷全冠对龈沟液内可溶性细胞间黏附分子-1及白细胞介素-1β水平的影响

目的 通过对不同内冠金属材料的烤瓷熔附金属(PFM)全冠修复后龈沟液(GCF)内可溶性细胞间黏附分子-1 (sICAM-1)及白细胞介素-1β(IL-1β)水平的测定,探讨内冠金属材料对牙周组织的影响.方法 将30颗牙分为镍铬合金组、钴铬合金组和金铂合金组,每组10颗,分别进行镍铬合金、钴铬合金和金铂合金PFM全冠修复...     

炎症人牙髓组织渗出液中IL-8含量的检测

目的：检测人正常和炎症牙髓组织渗出液中ＩＬ－８的含量,揭示ＩＬ－８与牙髓炎的关系。方法：用滤纸条浸润法采集正常和临床诊断为急性或慢性牙髓炎患牙牙髓组织渗出液。用ＥＬＩＳＡ方法检测其ＩＬ－８含量。结果：正常牙髓组织渗出液中检测不到ＩＬ－８,而炎症牙髓组织渗出液中均有较高水平的ＩＬ－８（０．３３～１．０μｇ／Ｌ）,且在急性牙髓炎中的水平高于慢性牙髓炎（Ｐ〈０．０１）。结论：ＩＬ－８参与了牙髓炎的发生和     

Intracellular complex carbohydrates in the normal and inflamed dental pulp

abstract — Forty-four inflamed pulps from human and monkey teeth were studied using histochemical staining methods. In pulps from carious human teeth, mast cells and many fibroblasts containing glycogen-like granules were regularly observed. In monkey pulps, where pulpitis was induced experimentally, mast cells were found only occasionally, and glycogen-like granules were absent. However, some fibroblasts in normal and inflamed monkey pulps contained periodic acid-Schiff (PAS) positive granules, which were resistant to amylase. Such granules were not found in human pulp fibroblasts.     

人正常及炎症牙髓中炎症信号受体TLR4表达的研究

目的:研究人正常及炎症牙髓组织中炎症信号受体TLR4的表达特点,探讨其在牙髓和根尖周组织炎症中的可能作用。方法:采用激光共聚焦扫描显微镜和免疫荧光技术,检测正常及炎症牙髓TLR4的表达情况。结果:正常牙髓组织中未发现TLR4免疫阳性细胞,炎症牙髓组织病灶处TLR4表达强阳性。正常组平均荧光强度为28.80±2.57,炎症组为258.12±24.51(P<0.001)。结论:与正常牙髓组织相比,炎症牙髓组织TLR4表达显著增强,提示其可能参与牙髓组织炎症过程。     

Ability of healthy and inflamed human dental pulp to reduce hydrogen peroxide

This study examined the defensive ability of human dental pulp against H2O2 in healthy and reversible and irreversible pulpitis tissues through determination of catalase activity by spectrophotometric methods. Thirty-five systemically healthy patients were donors of the pulp tissue, and pulp conditions were assessed using clinical and X-ray evaluations. Catalase activity was 1.61 +/- 0.23 U mg(-1) protein in the healthy tissues, 2.99 +/- 0.45 U mg(-1) protein in the reversible pulpitis tissues, and 2.44 +/- 467 mU mg(-1) protein in the irreversible pulpitis tissues. All differences between the groups were statistically significant. These results point to a role for catalase during dental pulp inflammation in humans, and therefore demonstrate an inherent biological defense system against reactive oxidants in human dental pulp.     

Up-regulation of nucleotide-binding oligomerization domain 1 in inflamed human dental pulp

Introduction

The innate immune response is activated by recognition of microbial components through specific pattern recognition receptors including nucleotide-binding oligomerization domain (NOD)-like receptors. However, the regulation of NOD-1 in inflamed human dental pulp remains poorly understood. This study aimed to evaluate the expression of NOD-1 in healthy and inflamed human dental pulps. In addition, the secretion of chemokines induced by NOD-1 and the related signaling pathways were studied.

Methods

Samples of human dental pulp tissues were obtained from freshly extracted wisdom teeth. The protein localization of NOD-1 in the pulp tissues was detected by immunohistochemistry. In addition, human dental pulp fibroblasts were stimulated with NOD-1 agonist γ-D-glutamylmeso-diaminopimelic acid. Production of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) was determined by an enzyme-linked immunosorbent assay. The involvement of mitogen-activated protein kinase (MAPK) signaling pathways was examined by Western blot analysis, and the association of MAPK signaling with chemokine production was determined.

Results

The results demonstrated the expression of NOD-1 in normal dental pulp, and up-regulated NOD-1 expression was observed in inflamed dental pulp. On stimulation with NOD-1 agonist, production of IL-8 and MCP-1 was induced in a dose-dependent manner. Moreover, phosphorylation of p38 MAPK and Jun N-terminal kinase (JNK) was enhanced by stimulation of NOD-1. With the treatment of p38 MAPK and JNK inhibitors, the NOD-1–induced IL-8 production was suppressed.

Conclusions

In response to microbial invasion, the expression of NOD-1 can be regulated in a ligand-inducible manner. NOD-1 might participate in pulp inflammation through chemokine production via MAPK signaling pathways.     

Immune components in normal and inflamed human dental pulp.

Normal and inflamed human pulpal tissues were examined for the presence of humoral immune components. The results indicate that normal pulpal tissue is essentially devoid of components necessary for localized immunoglobulin synthesis as shown by the absence of any immunoglobulin-containing cells (ICC). Tissue fluorescence was observed only in the unwashed samples and mainly with the fluoresceinated IgG reagent. This fluorescence was essentially eliminated by prewashing prior to the application of the fluoresceinated antiserums. On the other hand, inflamed pulpal tissues showed the presence of various ICC. IgG ICC were preponderant, constituting more than 60 per cent of ICC observed. Significant numbers of IgA and IgE ICC were also observed in each sample examined; whereas IgM ICC were present in only 3 of 12 specimens. At the tissue level, fluorescence was observed both in unwashed and prewashed tissues, predominantly with the labelled IgG antiserum. The presence of ICC, as well as extracellular immunoglobulin in inflamed pulpal tissue suggests that the potential exists for immune mechanisms to contribute to the pathological periapical changes seen as sequelae to pulpal inflammation.     

基质细胞衍生因子1在人炎症牙髓组织中表达的实验研究

目的:研究基质细胞衍生因子1(Stromal cell-derived factor-1,SDF-1)及其受体CXCR4在人炎性牙髓组织中的表达,探讨SDF-1/CXCR4轴在牙髓炎症发生发展中的可能作用。方法:采用免疫组织化学染色的方法检测SDF-1和CXCR4阳性细胞在健康、炎症牙髓组织中的分布情况。以实时荧光定量RT-PCR方法检测SDF-1mRNA在健康和炎症牙髓中的表达。结果:炎性牙髓中SDF-1、CXCR4主要分布于炎性细胞、成牙本质细胞和微血管内皮细胞。而正常组牙髓少见SDF-1、CXCR4阳性细胞。炎性牙髓中SDF-1mRNA的表达较健康牙髓显著增强。结论:与正常牙髓相比,炎性牙髓组织中SDF-1、CXCR4阳性细胞明显增多。炎性牙髓中SDF-1表达水平明显上调。SDF-1/CXCR4轴可能参与了牙髓炎症损伤和修复过程。     

人牙髓组织缺氧耐受机制的初步研究

目的：研究缺氧诱导因子-1α（Hypoxia inducible factor-1α,HIF-1α）和环氧化酶-2（Cyclooxygenase-2,COX-2）在人正常牙髓和炎症牙髓中的免疫定位,探讨缺氧耐受机制在牙髓炎病程中和牙髓自身修复过程中的作用和意义。方法：通过SP法对第1组10例健康牙髓、第2组10例深龋（有过敏症状但无牙髓炎症状）患牙牙髓、第3组15例急性牙髓炎牙髓和第4组15例慢性牙髓炎牙髓进行免疫组化染色,分别对HIF-1α和COX-2进行免疫定位和半定量分析。结果：①HlF-1α在第1组健康牙髓和第2组深龋牙髓标本中,只有个别标本呈弱阳性表达,二组问无显著性差异（P〉0．05）,但对比其它二组标本则有显著性差异（P〈0．001）：第3组急性牙髓炎牙髓呈强阳性表达,第四组慢性牙髓炎牙髓亦呈阳性染色与第3组比较总体偏弱,有显著性差异（P〈0．05）。②COX-2在第1组牙髓中个别标本呈弱阳性表达;第2组标本绝大多数染色呈弱阳性,与第1组比较有显著性差异（P〈0.05）;第3组标本染色均呈强阳性,与前两组比较有显著差异性伊值分别为P〈0.001,P〈0．05）;第4组标本亦呈阳性染色,总体上阳性程度明显高于第l组（P〈0．001）,高于第2组（P〈0．05）,弱于第3组（P〈0．05）。HIF-1α与COX-2的表达关系主要在第3组和第4组牙髓标本中体现明显,在第3组二者的变化大致呈平行关系;在第4组基本呈反向变化关系。结论：HIF-1α和COX-2在牙髓炎病程中可能发挥着重要的生理和病理作用,在牙髓炎进程中和牙髓自身修复过程中可能存在着缺氧环境和缺氧耐受机制。