内毒素对牙龈成纤维细胞mCD14表达的影响

目的:研究mCD14在牙龈成纤维细胞的膜表面分布及内毒素对mCD14在牙龈成纤维细胞膜表面表达的影响。方法:组织块法培养人牙龈成纤维细胞,利用免疫组化和Western blot方法研究mCD14在牙龈成纤维细胞的膜表面分布,同时观察内毒素对mCD14在牙龈成纤维细胞膜表面表达的影响。结果:免疫组织化学染色实验和蛋白印迹实验结果均表明mCD14在牙龈成纤维细胞的膜表面表达阳性,同时LPS可增强膜表面的mCD14的表达。结论:本实验表明正常人牙龈成纤维细胞可表达mCD14,LPS可以上调膜表面mCD14的表达,从而导致牙周损伤。     

牙周病龈组织中血栓素B2的放射免疫测定及免疫组化…

选取成人牙周炎、青少年牙周炎、龈炎和健康人各１０例，用放射免疫测定法测量龈组织中血栓素Ｂ２含量，用免疫组化ＡＢＣ染色对同一标本中ＴＸＢ２定位，用显微分光光度计对染色标本定量。结果表明：牙周病患者龈组织中ＴＸＢ２含量从健康人龈炎、青海年周炎到成人牙周炎呈递增趋势，且各组间均有显著性差异，ＴＸＢ２主要的分泌细胞为血管内皮细胞、上皮基底细胞，巨噬细胞和浆细胞，对牙龈组织中ＴＸＢ２的ＭＳＰ定量结果与放射免     

LPS刺激牙龈成纤维细胞IL-6、IL-8的产生和膜表面受体CD14的表达

目的：观察牙龈卟啉菌和中间普氏菌LPS刺激人牙龈成纤维细胞合成IL-6、IL-8的能力，并了解LPS是否通过细胞表面膜受体mCD14介导信号传导。方法：4种浓度的LPS在不同时间段。分别作用于体外培养的人牙龈成纤维细胞。采用ELISA检测IL-6、IL-8含量的变化，同时利用逆转录聚合酶链反应，进一步观察IL-6、IL-8，CD14在mRNA水平的表达特征。结果：ELISA和RT-PCR的反应结果证实，受LPS的刺激，细胞合成和分泌细胞因子IL-6、IL-8的能力明显增强，但未检测到细胞表面膜受体mCD14mRNA的表达。结论：LPS用于牙龈成纤维细胞合成和分泌细胞因子没有通过膜受体mCD14的介导。     

牙周病龈组织中血栓素B_2的放射免疫测定及免疫组化定位与定量分析

选取成人牙周炎、青少年牙周炎、龈炎和健康人各１０例，用放射免疫测定法测量龈组织中血栓素Ｂ２（ＴＸＢ２）含量，用免疫组化ＡＢＣ染色对同一标本中ＴＸＢ２定位，用显微分光光度计（ＭＳＰ）对染色标本定量。结果表明：牙周病患者龈组织中ＴＸＢ２含量从健康人龈炎、青少年牙周炎到成人牙周炎呈递增趋势，且各组间均有显著性差异。ＴＸＢ２主要的分泌细胞为血管内皮细胞、上皮基底细胞、巨噬细胞和浆细胞，对牙龈组织中ＴＸＢ２的ＭＳＰ定量结果与放射免疫测定结果相似，提示进一步开展ＴＸＢ２与牙周病之间关系的研究有重要意义。     

牙龈卟啉单胞菌的牙龈蛋白酶K-caspase样亚基的表达与青春期龈炎的临床相关性

目的检测并比较青春期龈炎的牙龈卟啉单胞菌（Porphyromonas gingivalis，PS）临床分离株的分泌蛋白和菌体蛋白中牙龈蛋白酶K（gingipain K，Kgp）-caspase样亚基的表达强度及酶活性，揭示Kgp与青春期龈炎之间可能存在的致病关系。方法检测并记录36例14～17岁的青春期龈炎患者的牙龈指数、龈沟出血指数及探诊深度，取龈下菌斑进行Pg的分离培养，16SrRNA PCR法鉴定。将获得的10个Pg临床分离株复苏，在对数生长期末提取分泌蛋白和菌体蛋白，测定其酶活性，并用Kgp-caspaae样亚基的单克隆抗体进行Western blot检测。Kgp的表达强度及酶活性与各牙周指数之间的相关关系用等级相关系数。结果青春期龈炎的Pg临床分离株的分泌蛋白和菌体蛋白中，Kgp-caspase样亚基的表达强度及酶活性与各牙周指数的大小呈正相关关系，差异有统计学意义（P〈0．05）。结论青春期龈炎龈下菌斑中Pg的Kgp十分复杂，存在表现为低相对分子质量形式的caapase样活性分子，这些细胞内功能蛋白分子将影响Pg和宿主间的相互作用；Kgp对青春期龈炎有一定的致病作用。     

增生性龈病损260例的组织病理学分析

龈增生是牙龈组织中细胞成分的增生和分化,其中最常见的是增生性龈炎,相对常见的有龈纤维瘤病和苯妥英龈增生。本研究观察了增生性龈炎、苯妥英龈增生、龈纤维瘤病的组织学,结合临床表现,着重探讨以下两个问题:1、龈增生的实质和发病机制;2、临床和病理上区别各种增生性龈病损。     

脂多糖对人牙髓细胞增殖和碱性磷酸酶活性的影响

目的:了解脂多糖(LPS)对人牙髓细胞的增殖和碱性磷酸酶活性的影响。方法:Ⅰ型胶原酶消化组织块法体外培养牙髓细胞,检测细胞表面LPS受体CD14(mCD14);流式细胞术观察不同浓度LPS(0.1μg/ml、1μg/ml和10μg/ml)对牙髓细胞细胞周期的影响,测定不同浓度LPS对牙髓细胞碱性磷酸酶活性的影响,采用χ2检验以及单因素方差分析对实验数据分别进行统计学分析。结果:人牙髓细胞不表达mCD14,LPS在浓度为0.1μg/ml、1μg/ml和10μg/ml时可以抑制牙髓细胞增殖(P<0.01)及碱性磷酸酶活性(P<0.05)。结论:LPS具有抑制牙髓细胞增殖及牙髓细胞分化的作用。     

根尖周炎动物模型中CD14、TLR4的表达

目的研究大鼠根尖周炎炎症组织中脂多糖炎症信号受体CD14、Toll样受体4（Toll．1ikereceptor4，TLR4）的表达特点，探讨根尖周炎中脂多糖的信号转导途径。方法建立大鼠磨牙内毒素根尖周炎模型，采用免疫组化染色观察根尖周炎炎症组织中CD14、TLR4的表达情况，并计算CD14、TLR4的阳性细胞率。结果正常根尖周组织中未发现CD14和TLR4免疫阳性细胞，根尖周炎症组织中CD14和TLR4表达阳性，CD14、TLR4阳性细胞率差异无统计学意义（P〉0．05）。结论与正常根尖周组织相比，炎症根尖周组织中CD14和TLR4的表达显著增强，CD14和TLR4的表达量差异无统计学意义，提示脂多糖可能通过CD14、TLR4信号受体在根尖周炎症中发挥作用。     

新生牛牙龈来源细胞的成骨特性

目的 在不同培养条件下，观察新生牛牙龈组织中是否含有具成骨细胞表型特征的细胞。方法 采用体外组织块法培养新生牛牙龈组织来源的细胞，根据加矿化液与否分为两组，并用免疫细胞（组织）化学及体外矿化实验观察其生物学特性。结果 牙龈结缔组织来源细胞在条件培养基中体外培养14天后，骨桥蛋白，骨涎蛋白，骨粘结素及骨钙素等与矿化相关的非胶原蛋白表达阳性；碱性磷酸酶表达亦呈阳性；而且有散在矿化小节形成，结论 新生牛骨龈结缔组织中的一些细胞在一定培养环境中可沿成骨细胞方向分化。具有成骨细胞的特性。     

龈炎和牙周炎时龈的微血管和浸润细胞的变化

在为慢性卡他性龈炎和慢性广泛性牙周炎施行手术时切取龈组织块44例,进行组织化学检查:测定龈微血管壁的ATP酶和碱性磷酸酶活性,用细胞光度计评定组织化学反应强度,光密度数据经电算机处理。龈组织形态学检查,计算龈间质单位面积的微血管数和     

Involvement of vascular endothelial growth factor, CD44 and CD133 in periodontal disease and diabetes: an immunohistochemical study

Aim: The aim of this study was to investigate the relationship between expression of angiogenic and regeneration markers and periodontal disease in subjects with/without diabetes mellitus.
Material and Methods: Immunohistochemical detection of vascular endothelial growth factor (VEGF), CD44 and CD133 was performed in 16 samples each of (1) healthy gingiva from non-diabetic subjects (controls), (2) gingiva from non-diabetic subjects with periodontitis, (3) gingiva from subjects with type 1 diabetes and periodontitis, (4) gingiva from subjects with type 2 diabetes and periodontitis.
Results: Diseased gingivae from patients with diabetes and periodontitis had greater clinical measures of periodontal disease than those with periodontitis only. VEGF expression was significantly enhanced in epithelial and endothelial cells from patients with periodontitis compared with controls ( p <0.05). Epithelial CD44 expression was strong in all groups, while CD44 was significantly enhanced ( p <0.05) in connective tissue cells from both diabetic groups. Epithelial and endothelial CD133 expression was comparable in all patients except those with type 2 diabetes and periodontitis, where it was not detected. Stromal CD133 expression was significantly lower in patients with type 2 diabetes and periodontitis and was increased in periodontitis patients ( p <0.05).
Conclusions: The involvement and high expression of VEGF, CD44 and CD133 in periodontal disease may predict a greater regeneration capacity of gingival tissue.     

Immunohistochemical localization of Toll-like receptors 2 and 4 in gingival tissue from patients with periodontitis

The present study investigated the expression of Toll-like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2-, TLR4-, CD14- and CD1a-positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2-positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4-positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14-positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down-regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.     

Immunohistochemical localization of Toll‐like receptors 2 and 4 in gingival tissue from patients with periodontitis

The present study investigated the expression of Toll‐like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2‐, TLR4‐, CD14‐ and CD1a‐positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2‐positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4‐positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14‐positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down‐regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.     

脂多糖信号受体CD14和 TLR4在人炎症根尖周组织中的表达

目的 研究慢性根尖周炎患者根尖周组织中脂多糖(LPS)信号受体CD14和TLR4的表达和分布,探讨CD14和TLR4在炎性根尖周组织中可能的作用.方法 收集需做根尖外科手术的12例慢性根尖周炎患者和10例外科手术拔出的无牙髓炎和根尖周病变的阻生齿的根尖周组织,术中取炎症和正常根尖周组织后常规切片,采用免疫组织化学方法检测CD14和TLR4的表达和分布.结果 CD14和TLR4在炎症根尖周组织中呈强阳性表达,主要分布于单核细胞、巨噬细胞等炎症细胞;而在正常根尖周组织中未见明显CD14和TLR4阳性细胞.结论 炎症根尖周组织中CD14和TLR4的阳性表达,提示LPS可能通过CD14和 TLR4信号受体在根尖周炎症组织中发挥作用.     

Toll like receptor 4 and membrane-bound CD14 expressions in gingivitis, periodontitis and CsA-induced gingival overgrowth

Objective

Immune cell recognition of lipopolysaccharides via CD14 and Toll-like receptor 4 (TLR4) complexes plays a crucial role in linking innate and adaptive immune responses. This study was aimed to investigate the expression of TLR4 and membrane-bound CD14 (mCD14) in the gingival tissues of patients with gingivitis, periodontitis and CsA-induced gingival overgrowth.

Design

Gingival tissues were obtained from 10 renal transplant patients receiving cyclosporine-A (CsA) and having gingival overgrowth (GO), 10 patients with chronic periodontitis, 10 generalized aggressive periodontitis, 10 gingivitis and 10 healthy subjects. Immunohistochemistry was performed in order to determine the localization of TLR4 and mCD14 in tissue specimens.

Results

TLR4 and mCD14 expressions were detected in all tissues including healthy gingival biopsies. TLR4 and mCD14 positive cells were predominantly confined to the epithelium–connective tissue interface area, and were highly expressed in the basal cell layer of patients with CsA GO and chronic periodontitis, compared to healthy group (P < 0.05).

Conclusion

The present study suggests that TLR4 and mCD14 protein expressions may be interrelated and appear to be associated with periodontal disease. CsA usage seemed not to affect TLR4 and mCD14 expressions in CsA induced GO tissues.     

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.     

In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA-expressing cells in human inflamed gingival tissue

This study presents the exact cell types and localization of tissue inhibitors of metalloproteinases (TIMPs) production sites in periodontal diseased gingiva by means of in situ hybridization. Gingival tissue specimens were fixed, embedded and hybridized in situ with specific digoxigenin-labeled cRNA probes (386 and 496 bp). TIMP-1 and -2 mRNAs were expressed on macrophages, mononuclear cells, capillary endothelial cells and some fibroblasts throughout the gingival tissue. In periodontitis, TIMP-1 and -2 mRNA-expressing cells showed significantly different localization. TIMP-1 mRNA was broadly observed in the gingival connective tissue while TIMP-2 mRNA was predominantly expressed in the connective tissue adjacent to the pocket epithelium (p < 0.01). Fewer TIMPs mRNA were observed in minimal gingivitis than in periodontitis, especially in the middle zone of gingival tissue. Thus, TIMP-1 and TIMP-2 mRNA was detected differentially and site-specifically in periodontal diseased gingival tissue.     

CD9 and HLA-DR expression by crevicular epithelial cells and polymorphonuclear neutrophils in periodontal disease

BACKGROUND, AIMS: The composition of gingival crevicular fluid (GCF) is likely to reflect inflammatory modifications that take place in the gingiva during periodontal diseases. METHOD: In this study, GCF was collected at 3 different sites from 23 periodontal patients. The sites were assessed to be healthy, presenting gingivitis or periodontitis. 10 healthy individuals without any form of periodontal disease formed the control group and were sampled at one site each. The cell content of GCF was collected using Durapore Millipore strips, and 2 types of cells were studied: epithelial cells (EC) and polymorphonuclear neutrophils (PMN). The expression of CD9 and HLA-DR within or on the surface of these cells was studied in immunofluorescence on cytospin smears. RESULTS: Both CD9 and HLA-DR expression on EC differed significantly from control subjects, and the latter decreased according to the severity of the pathology. None of the PMN found in controls expressed CD9 or HLA-DR. However, in periodontal patients, the expression of HLA-DR within PMNs was detectable and increased according to the severity of lesions. CD9 expression on PMNs also increased with inflammation. CONCLUSION: This study shows that clinically healthy sites of periodontal patients already present signs of immunological activation characterised by a down modulation of HLA-DR expression on EC and an upregulation of these 2 molecules in PMN.     

The defensive role of lysozyme in human gingiva in inflammatory periodontal disease

Background and Objective:  The presence of lysozyme in human gingiva has not previously been demonstrated. In this study, we looked for evidence for the potential role of lysozyme as a protector of gingival elastic fibres. The objective of this study was also to determine the ex vivo susceptibility to hydrolysis of gingival elastic fibres from patients with or without periodontal disease by human leukocyte elastase and by human cathepsin G.
Materials and Methods:  Using gingival tissue sections from eight control, 10 gingivitis and 10 periodontitis patients, we evaluated the area fraction occupied by gingival elastic fibres (after selective staining) by the use of automated image analysis. In the ex vivo experiments, serial tissue sections from four control, four gingivitis, four young periodontitis and four aged periodontitis patients were submitted to the action of human leukocyte elastase and cathepsin G, after which enzymatic activities were determined by image analysis. Indirect immunodetection of lysozyme was also done on tissue sections for all patients included in this study.
Results:  Large variations of the area fraction occupied by elastic fibres were observed in human gingiva from young and aged patients with and without periodontal disease. In control and gingivitis patients, leukocyte elastase and cathepsin G had high comparable elastin solubilizing activities. With young and aged periodontitis patients, the two serine proteinases had weak elastin solubilizing activities. Lysozyme appeared to be present at the periphery of gingival elastic fibres in periodontitis patients.
Conclusion:  Lysozyme can be considered an important natural protector of elastic fibres in pathological gingiva.     

Increased infiltration of CD1d and natural killer T cells in periodontal disease tissues

BACKGROUND: Natural killer T (NKT) cells are a unique T lymphocyte subset that has been implicated in the regulation of immune responses associated with a broad range of diseases including autoimmunity, infectious diseases, and cancer. In contrast to conventional T cells, NKT cells are reactive to major histocompatibility complex (MHC) class I-like molecule CD1d. Considering the periodontitis having both aspects of infection and autoimmunity in nature, CD1d and reactive NKT cells are of particular importance. OBJECTIVE: The aim of the present study was to examine whether the expression of CD1 isoforms and Valpha24(+) invariant NKT cells is associated with different disease entities, namely gingivitis and periodontitis. MATERIAL AND METHODS: Immunohistochemical analysis was performed on cryostat sections of gingival tissues from 19 patients with periodontitis and eight patients with gingivitis using antibodies to CD1a, b, c, d, Valpha24(+) invariant NKT cells, CD83, CD3 and CD19. RESULTS: Although all four subsets of CD1 molecules were expressed in periodontal lesions, CD1d was most abundant. CD1d expression was more frequent in periodontitis than gingivitis and increased together with increase of invariant NKT cell infiltration. Double immunohistochemical staining showed co-expression of CD1d and CD19 on identical cells and proximate infiltration of CD1d(+) and invariant NKT cells. CONCLUSION: These findings suggest that CD1d-expressing B cells could activate NKT cells by CD1d-restricted manner and this NKT cell activation may play roles in pathogenesis of periodontal diseases.