老年牙周炎牙龈组织诱导型一氧化氮合酶分布研究

目的 :观察慢性老年牙周炎患者牙龈组织中诱导型一氧化氮合酶 (iNOS)分布。方法 :采用免疫组织化学方法对 10例慢性老年牙周炎患者、10例慢性成人牙周炎患者、10例青少年牙周炎患者和 10例健康老年人牙龈组织中诱导型一氧化氮合酶分布进行了检测并比较研究。结果 :(1)牙周炎时牙龈组织中诱导型一氧化氮合酶主要在鳞状上皮细胞胞浆核周区颗粒状阳性表达 ,毛细血管壁内皮细胞、老化的胶原纤维及上皮下基底膜共同形成了一种乳头状轮廓样阳性表达形态 ,结缔组织和肉芽组织中各类炎症细胞也显阳性表达 ;(2 )慢性老年牙周炎组血管壁内皮细胞、结缔组织内炎症细胞、上皮乳头阳性表达例数明显低于青少年牙周炎组和慢性成人牙周炎组 (P <0 .0 5 )。血管壁内皮细胞和胶原纤维阳性表达例数低于健康老年人组 (P <0 .0 5 )。结论 :慢性老年牙周炎患者牙龈组织中诱导型一氧化氮合酶的表达明显降低 ,造成了局部一氧化氮(NO)合成减少 ,引起了局部牙龈组织免疫功能降低和免疫调节功能紊乱     

血管内皮生长因子在正常与牙周炎牙龈组织中的表达差异研究

目的 检测血管内皮生长因子(VEGF)在正常与牙周炎的牙龈组织中的表达差异。方法 采用免疫组化PV两步法,检测VEGF在20例健康成人与20例慢性中重度牙周炎患者的牙龈组织中的表达差异。结果 健康组牙龈组织VEGF表达局限于上皮的颗粒层与部分棘层,其下方的结缔组织呈弱阳性表达,实验组牙龈组织上皮全层及结缔组织均呈强阳性表达,实验组VEGF表达高于健康组,两组之间的差异具有统计学意义(上皮区P<0.01,结缔组织区P<0.05)。结论 VEGF可能参与了牙周炎牙周袋壁龈组织的病理改变过程,在牙周炎发生发展及修复机制中可能具有重要意义。     

细胞粘附分子ICAM-1在慢性牙周炎牙龈组织中白细胞的表达

目的　研究ICAM - 1在慢性牙周炎牙龈组织中白细胞的表达 ,探讨ICAM - 1在慢性牙周炎免疫病因机理中和作用。方法　应用抗ICAM - 1单克隆抗体 ,通过碱性磷酸酶 -抗碱性磷酸酶的免疫组织化学技术 ,对I CAM - 1在正常和慢性牙周炎的牙龈组织中白细胞的表达进行分析。结果　ICAM - 1阳性白细胞主要聚集于慢性牙周炎结合上皮和袋上皮下方结缔组织中 ,慢性牙周炎牙龈组织单位面积中表达ICAM - 1阳性的白细胞的比例显著高于正常牙龈组 (P <0 .0 1)。结论　ICAM - 1可能参与和调节慢性牙周炎牙龈组织中白细胞介导的免疫粘附过程。     

细胞粘附ICAM-1慢性牙周炎牙龈组织中白细胞的表达

目的研究ICAM-1在慢性牙周炎牙龈组织中白细胞的表达,探讨ICAM-1在慢性牙周炎免疫病因机理中和作用.方法应用抗ICAM-1单克隆抗体,通过碱性磷酸酶-抗碱性磷酸酶的免疫组织化学技术,对ICAM-1在正常和慢性牙周炎的牙龈组织中白细胞的表达进行分析.结果 ICAM-1阳性白细胞主要聚集于慢性牙周炎结合上皮和袋上皮下方结缔组织中,慢性牙周炎牙龈组织单位面积中表达ICAM-1阳性的白细胞的比例显著高于正常牙龈组(P＜0.01).结论 ICAM-1可能参与和调节慢性牙周炎牙龈组织中白细胞介导的免疫粘附过程.     

β-防御素-2多肽在不同类型牙周炎及健康牙龈组织中的表达

目的:检测人β-防御素-2(humanbetadefensin-2,HBD-2)在牙周病病变牙龈和健康牙龈中的表达。方法:应用SP法检测健康牙龈(HC组,11例)、慢性牙周炎(CP组,18例)和侵袭性牙周炎(AgP组,9例)牙龈中HBD-2蛋白的表达水平,所得数据用SPSS11.5统计分析软件进行单因素方差分析。结果:HBD-2蛋白在所有牙龈标本中均有表达;HBD-2蛋白表达可见于牙龈复层鳞状上皮全层,主要位于棘层以上细胞胞质内。3组的表达水平分别为:健康牙龈组(31.55±12.66)%、慢性牙周炎组(17.31±7.64)%、侵袭性牙周炎组(25.06±8.04)%,健康组表达水平显著高于慢性牙周炎组(P<0.05),其他组间差异无统计学意义。结论:HBD-2多肽在健康牙龈和牙周病牙龈上皮中广泛表达,提示其在维系牙周健康及宿主免疫防御反应中可能发挥重要作用。     

细胞间粘附分子1及其受体在成人牙周炎袋上皮区的表达

目的 探讨成人牙周炎牙龈组织中白细胞通过上皮进入龈沟液的粘附机制。方法 采用碱性磷酸酶－抗碱性磷酸酶免疫组化方法，对细胞间粘附分子／白细胞功能相关抗原１在正常牙龈和成人牙周炎牙龈组织中的表达进行研究。结果 正常牙龈和成人牙周炎牙龈的结合上皮，沟内／袋上皮根部均表达ＩＣＡＭ－１，且染色强度呈方向变化；ＬＦＡ－１仅局限表达于白细胞表面，成人牙周炎袋上皮下方结缔组织及袋上皮细胞间隙中ＬＦＡ－１阳性白细胞     

老年人牙周炎牙龈组织中凋亡细胞免疫组织化学研究

目的 :观察老年人牙周炎牙龈组织中凋亡细胞分布特点。方法 :采用特异性凋亡细胞免疫组织化学染色法 (TUNEL染色 ) ,对 15例老年人牙周炎患者 ,10例慢性成人牙周炎患者 ,10例青少年牙周炎患者和 11例健康老年人牙龈组织中阳性凋亡细胞的分布及计数进行比较研究。结果 :老年人牙周炎组阳性凋亡细胞总例数明显高于健康老年人组和慢性成人牙周炎组 (P <0 .0 5 ) ;慢性成人牙周炎组和青少年牙周炎组阳性凋亡细胞总例数也明显高于健康老年人组 (P <0 .0 5 ) ;老年人牙周炎组阳性凋亡细胞计数明显高于慢性成人牙周炎组、青少年牙周炎组和健康老年人组 (P <0 .0 5 )。结论 :感染性炎症因素和衰老因素均能促进老年人牙周炎时牙龈组织细胞凋亡 ,这两个因素的双重协同作用造成了老年人牙周炎患者局部牙龈组织对刺激做出过强的细胞凋亡反应。     

年龄对NK细胞在牙周炎龈组织中分布影响的免疫组化研究

目的：观察老年牙周炎牙龈组织中自然杀伤（NK）细胞的分布。方法：采用免疫组织化学方法对15例老年牙周炎患者10例慢性成人牙周炎患者牙龈组织中NK细胞、T淋巴细胞和B淋巴细胞的分布及阳性计数进行比较研究。结果：慢性成人牙周炎组NK阳性细胞计数明显高于老年牙周炎组（P＜0.01)，慢性成人牙周炎组NK阳性细胞表达率也高于老年牙周炎组。结论：由于年龄增加的影响，老年牙周炎患者局部牙龈组织中出向免疫调节功能的紊乱，NK细胞功能降低。     

慢性牙周炎患者牙龈组织内诱导型一氧化氮合酶表达的研究

目的:研究牙周健康者和慢性牙周炎患者牙龈组织中诱导型一氧化氮合酶的表达强度,探讨一氧化氮在牙周病发病过程中的作用.方法:选择牙周健康组、慢性牙周炎活动期组,慢性牙周炎静止期组各20例,采取免疫组织化学的方法染色,光镜下观察牙龈组织内诱导型一氧化氮合酶的表达强度.结果:慢性牙周炎时牙龈组织中诱导型一氧化氮合酶主要在鳞状上皮和间质组织的细胞胞浆中阳性表达,正常组表达强度弱于慢性牙周炎静止期组和活动期组,慢性牙周炎静止期组表达强度弱于慢性牙周炎活动期组.结论:一氧化氮参与了慢性牙周炎的发生和发展过程,牙龈组织中诱导型一氧化氮合酶的表达强度与慢性牙周炎的炎症程度密切相关.     

基质金属蛋白酶-2在人慢性牙周炎肥大细胞上表达的研究

目的观察基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)在慢性牙周炎牙龈组织中肥大细胞上的表达,探讨MMP-2-tryptase双阳性肥大细胞(mast cells,MCs)在慢性牙周炎发病机制中的作用。方法将45例参试者依据慢性牙周炎的病变程度分成3组:健康对照组15例;轻度牙周炎组15例;重度牙周炎组15例。牙龈标本经10%福尔马林液固定48 h;制作牙龈组织连续切片,HE染色,光学显微镜下观察组织学改变;采用免疫荧光双染色法,荧光显微镜下观察牙龈组织中MMP-2-tryptase双阳性MCs的表达情况。结果与健康对照组相比,轻度和重度慢性牙周炎组牙龈组织MMP-2-tryptase双阳性MCs密度均显著升高(P<0.01);重度慢性牙周炎组牙龈组织MMP-2-tryptase双阳性MCs密度显著高于轻度牙周炎组(P<0.05)。结论慢性牙周炎牙龈组织MMP-2-tryptase双阳性MCs密度与牙周炎病变程度的趋势相一致,MMP-2-tryptase双阳性MCs在牙周炎的进展中可能有促进作用。     

Immunohistochemical localization of CD1a and S100 in gingival tissues of healthy and chronic periodontitis subjects

Oral Diseases (2012) 18, 778-785 Objective: The aim of this study was to evaluate the presence and distribution of CD1a and S100 protein markers in states of gingival health and chronic periodontitis in human subjects. Materials and Methods: Gingival tissue samples were derived from 10 healthy and 10 chronic periodontitis-affected human subjects. The presence and distribution of CD1a and S100 protein was assessed using immunohistochemistry, and the cell types involved in their expression was determined. Results: The presence and distribution of CD1a was confined only to the gingival epithelium, whereas S100 was seen in the epithelium and connective tissue. However, increased expression of both CD1a and S100 protein was seen in periodontitis-affected gingival tissues compared with healthy gingiva. Immunohistochemistry demonstrated that CD1a- and S100-positive cells in the epithelium are Langerhans cells (LCs) and S100 positive cells in the connective tissue are dendritic cells (DCs). Conclusion: Our findings suggest the transition of CD1a-positive LCs to S100-positive DCs from epithelium to connective tissue in response to an antigenic challenge. Demonstration of increased number of S100-positive DCs in the gingival connective tissue in chronic periodontitis possibly suggests their involvement in bone resorption in addition to their antigen presentation property.     

Expression of ICAM-1/LFA-1 in the pocket area of adult periodontitis]

OBJECTIVE: To study the expression of ICAM-1/LFA-1 in the pocket area of adult periodontitis. METHODS: Expressions of ICAM-1/LFA-1 in adult gingival of periodontitis and healthy subjects were studied by alkaline phosphorylase-antialkaline phosphorylase technique. RESULTS: Junctional epithelium and apical part of sulcus epithelium expressed ICAM-1 in both adult periodontitis and healthy gingiva, showing an ICAM-1 gradient change with maximal staining at tooth aspect and weaker staining in the basal layer of keratinocytes. Significantly more LFA-1 positive leukocytes were observed in connective tissues and within pocket epithelium in adult periodontitis than those in healthy gingiva. CONCLUSION: ICAM-1/LFA-1 may provide important adhesion pathway for leukocytes migration into gingiva sulcus in adult periodontitis lesions.     

Memory T cell subsets in healthy gingiva and periodontitis tissues

1 Background

In the gingival sulcus, effective and balanced innate and adaptive immune responses against subgingival plaque microbiome are crucial to maintain immune homeostasis. In this study, we investigated the memory T cell subsets in healthy gingiva and periodontitis tissues.

2 Methods

Anatomical localization of T cells (CD3⁺, CD4⁺, and CD8⁺) in healthy gingiva and periodontitis tissues were examined immunohistochemically. Subsets of memory T cells from isolated gingival cells were analyzed by flow cytometry using a cocktail of monoclonal antibodies (anti‐CD69, anti‐CD103, anti‐CD45RA, anti‐CCR7, anti‐CD28, and anti‐CD95). Intracellular cytokine staining of interleukin (IL)‐17 and interferon (IFN)‐γ expression on memory T cells in periodontitis tissues was also investigated.

3 Results

We found that healthy gingiva contains two memory T cell populations; a CD69^? recirculating population and a CD69⁺ gingiva‐resident memory T cell population. CD4⁺ T cells with transitional memory (T_TM) phenotype (CD45RA^?CCR7^?CD28⁺CD95⁺) constitute the major subset within these two populations. A significant increase in the proportion of CD4⁺CD69⁺CD103^? memory T cells was observed in periodontitis tissues compared with healthy gingiva. CD4⁺ memory T cells from periodontitis tissues produced either IL‐17 or IFN‐γ whereas CD8⁺ memory T cells produced only IFN‐γ.

4 Conclusions

Our findings suggest that recirculating and gingiva‐resident memory T cells could represent an important part of the immune surveillance network in the connective tissue, maintaining periodontal homeostasis. Imbalance of subgingival bacterial communities could damage gingival barrier allowing bacterial antigens to get access to the deeper connective tissue where they activate memory T cells leading to deleterious inflammation; a hallmark of periodontitis.     

纤维调节素在大鼠和人类牙龈组织中的差异性表达

目的:研究纤维调节素在大鼠和人类健康和牙龈组织中的分布与表达。方法:用免疫组织化学的方法研究纤维调节素在大鼠牙龈及人类健康和炎性牙龈组织中的分布。与之比较,I型胶原纤维在鼠磨牙牙龈组织中的分布也做了研究。结果:在大鼠牙龈中,纤维调节素分布在牙龈上皮基底上层,在腭侧牙龈结缔组织有强表达。I型胶原纤维也有类似的分布。比较而言,纤维调节素在颊侧牙龈结缔组织表达较弱。对于人类,纤维调节素在炎性牙龈结缔组织中表达明显,然而在健康牙龈结缔组织中表达较弱。结论:与Ⅰ型胶原纤维关系密切的纤维调节素在颊侧和腭侧牙龈中有差异性表达,并且在人类炎性牙龈组织中表达增强。     

The presence of bacteria in the oral epithelium in periodontal disease. II. Immunohistochemical identification of bacteria

Serial histological sections of gingiva obtained from each of six advanced adult periodontitis, two localized juvenile periodontitis and two periodontally healthy patients were used for specific identification of bacteria within the oral epithelium and adjacent connective tissue. Healthy gingival biopsies served as controls. Sections from patients and control biopsies were Gram-stained and also screened with antibacterial sera associated with the peroxidase immunocytochemical technique for specific bacterial identification. The "Pop-off" electron microscopic technique was also used to further demonstrate the bacterial nature of peroxidase-stained material. In addition, the possible correlation between bacteria and areas of possible reduced keratinization was investigated. The results showed that sections of orthokeratinized healthy gingiva did not contain bacteria. Gram-stained sections from diseased sites contained large numbers of bacteria in the oral epithelium and adjacent connective tissue. Bacteroides gingivalis and to a lesser extent Capnocytophaga gingivalis were found in periodontitis, and Actinobacillus actinomycetemcomitans was found in juvenile periodontitis when the immunoperoxidase technique was used. The bacterial nature of peroxidase-stained material was confirmed by the "pop-off" technique. In the disease biopsies, bacterial presence was correlated with areas of reduced amounts of keratin suggesting that the oral epithelium may be a portal of entry for bacteria into gingival tissues.     

Polymorphonuclear neutrophils and their mediators in gingival tissues from generalized aggressive periodontitis.

BACKGROUND: Impaired polymorphonuclear neutrophil (PMN) functions were generally considered to be related to the onset of generalized aggressive periodontitis (GAgP). However, some research has indicated that the hyperreactivity of PMN seems to be involved in the inflammatory response of GAgP. The present study's main purpose was to provide more evidence about the role of PMN in the pathogenesis of GAgP by surveying PMN infiltration in gingiva and its relationship with the expression of their mediators including intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). The inflammatory response in GAgP was also compared with that in adult periodontitis (AP) and periodontally healthy subjects. Since these PMN mediators were reported to be produced mainly by macrophages, the association between the expression of these PMN mediators and the distribution of macrophages was also investigated. METHODS: A total of 25 gingival specimens were obtained from 10 GAgP patients, 10 AP patients, and 5 periodontally healthy subjects. Serial sections were obtained from each specimen, and the following techniques were adopted to investigate the distribution and interrelation of different cells and cytokines. Infiltration of PMN was observed by using hematoxylin and eosin staining. Distribution of the macrophages, identified as CD68+, was shown by using immunohistochemistry. Immunohistochemistry and in situ hybridization were used to detect the expression of ICAM-1, IL-8, IL-1beta, and TNF-alpha in gingival tissues. These techniques were performed in serial sections from each individual specimen. RESULTS: Large numbers of infiltrating PMNs were observed in gingiva from GAgP. In gingiva from both GAgP and AP, the strongest protein and mRNA expression of IL-8, ICAM-1, IL-1beta, and TNF-alpha were located in pocket epithelium and adjacent connective tissue with large numbers of infiltrating PMNs. In tissues without abundant PMN infiltration, the appearance of positive cells expressing IL-8, ICAM-1, IL-1beta, and TNF-alpha was scattered. CD68+ was distributed sparsely in connective tissue and was hardly seen in pocket epithelium with large numbers of PMN infiltration. The degree of leukocyte infiltration and connective tissue destruction in gingiva from GAgP patients was not distinctly different from that in gingiva from AP. The gingival specimens with heavy PMN infiltration from both GAgP and AP patients presented strong expressions of IL-1beta and TNF-alpha; showed more extensive inflammatory cell infiltration; had severe connective tissue destruction; and presented severe elongation and ulceration of pocket epithelium. In gingiva from healthy subjects, inflammation was minor with visually no PMN, CD68+, or the positive cells of IL-8, ICAM-1, IL-1beta and TNF-alpha expression. CONCLUSIONS: Enhanced accumulation of PMN, which is associated with the upregulation of IL-8, ICAM-1, IL-1beta, and TNF-alpha expression, relates to the severity and activity of GAgP. In addition to macrophages, PMN and/or epithelial cells might also be important sources of IL-8, IL-1beta, and TNF-alpha production in gingiva.     

尼古丁对人牙周膜成纤维细胞中Fas/Apo-1（CD95）表达的影响

目的：研究尼古丁（nicotine）对体外培养的人牙周膜成纤维细胞（periodontal ligament fibroblasts,PDLFs）中Fas/Apo-1（CD95）表达的影响。方法：采用组织块培养法培养原代人牙周膜成纤维细胞并传代取处于对数生长期的第6代细胞用于实验,采用浓度为2mg/L的尼古丁处理第6代牙周膜成纤维细胞72h,通过免疫组织化学染色法检测尼古丁对人牙周膜成纤维细胞中Fas表达的情况。结果：证实尼古丁作用于人牙周膜成纤维细胞后,胞膜中Fas/Apo-1（CD95）抗原呈阳性表达。结论：提示尼古丁可通过Fas系统这一途径导致人牙周膜成纤维细胞的凋亡,从而加重牙周组织破坏的程度和影响牙周组织的再生。     

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.