慢性牙周炎龈组织中Smac和Bax的表达及其对细胞凋亡的作用

目的:通过检测Smac和Bax蛋白在慢性牙周炎龈组织中的表达,旨在探讨二者的相关性及其在细胞凋亡中与牙周炎发生、发展的关系。方法:慢性牙周炎测试组27人,健康对照组27人,利用光镜和透射电镜观察凋亡细胞形态结构,流式细胞仪测定线粒体膜电位。免疫组化检测Smac、Bax在龈组织不同区域的表达。结果:光镜观察慢性牙周炎龈组织中存在有凋亡细胞;电镜观察凋亡细胞中线粒体形态结构改变;慢性牙周炎龈组织线粒体膜电位降低,与健康组比较,两组间有显著性差异(P<0.05);慢性牙周炎组中Smac和Bax在上皮组织、结缔组织的表达增加,与健康组相比具有显著性差异(P<0.05);Smac、Bax二者的表达在慢性牙周炎龈组织具有明显正相关(P<0.05)。结论:慢性牙周炎龈组织中有凋亡细胞存在;Smac、Bax的表达在慢性牙周炎组中呈明显正相关,表明在细胞凋亡过程中起协同作用,这与牙周组织破坏有关。     

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。     

内质网应激诱导的细胞凋亡在慢性牙周炎中的作用初探

目的:初步探讨内质网应激诱导的细胞凋亡在慢性牙周炎中的作用。方法:利用光学显微镜和透射电镜观察牙周组织中的凋亡细胞,免疫组化染色法检测慢性牙周炎患者及牙周健康者牙周组织中GRP78和CHOP的分布及含量变化。结果:慢性牙周炎组内浆细胞呈现凋亡状态,且GRP78和CHOP在慢性牙周炎组中的阳性表达明显高于正常组(P<0.05)。结论:内质网应激参与了慢性牙周炎的病理生理过程。     

Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis-affected gingival tissues

Abe D, Kubota T, Morozumi T, Shimizu T, Nakasone N, Itagaki M, Yoshie H. Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis‐affected gingival tissues. J Periodont Res 2011; 46: 345–353. © 2011 John Wiley & Sons A/S Background and Objective: Gene expression is related to the pathogenesis of periodontitis and plays a crucial role in local tissue destruction and disease susceptibility. The aims of the present study were to identify the expression of specific genes and biological pathways in periodontitis‐affected gingival tissue using microarray and quantitative real‐time RT‐PCR analyses. Material and Methods: Healthy and periodontitis‐affected gingival tissues were taken from three patients with severe chronic periodontitis. Total RNAs from six gingival tissue samples were used for microarray analyses. Data‐mining analyses, such as comparisons, gene ontology and pathway analyses, were performed and biological pathways with a significant role in periodontitis were identified. In addition, quantitative real‐time RT‐PCR analysis was performed on samples obtained from 14 patients with chronic periodontitis and from 14 healthy individuals in order to confirm the results of the pathway analysis. Results: Comparison analyses found 15 up‐regulated and 13 down‐regulated genes (all of which showed a change of more than twofold in expression levels) in periodontitis‐affected gingival tissues. Pathway analysis identified 15 up‐regulated biological pathways, including leukocyte transendothelial migration, and five down‐regulated pathways, including cell communication. Quantitative real‐time RT‐PCR verified that five genes in the leukocyte transendothelial migration pathway were significantly up‐regulated, and four genes in the cell communication pathway were significantly down‐regulated, which was consistent with pathway analysis. Conclusion: We identified up‐regulated genes (ITGB‐2, MMP‐2, CXCL‐12, CXCR‐4 and Rac‐2) and down‐regulated genes (connexin, DSG‐1, DSC‐1 and nestin) in periodontitis‐affected gingival tissues; these genes may be related to the stimulation of leukocyte transendothelial migration and to the the impairment of cell‐to‐cell communication in periodontitis.     

老年牙周炎牙龈组织Fas/Apo-1(CD95)抗原表达的免疫组织化学研究

目的 :观察慢性老年牙周炎牙龈组织中Fas/Apo - 1(CD95 )抗原表达和分布情况。方法 :采用免疫组织化学染色方法对 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Fas/Apo - 1(CD95 )抗原阳性表达和分布情况进行了观察和比较。结果 :在慢性老年牙周炎组完整的牙龈鳞状上皮间桥、核周胞浆Fas/Apo - 1(CD95 )抗原阳性表达和结缔组织中淋巴细胞Fas/Apo - 1(CD95 )抗原阳性表达为 4组中最强 ;健康老年人组上皮角化层Fas/Apo - 1(CD95 )抗原阳性表达为 4组中最强 ;慢性成人牙周炎组和青少年牙周炎组结缔组织中淋巴细胞和吞噬细胞Fas/Apo - 1(CD95 )抗原阳性表达强于健康老年人组 ;慢性老年牙周炎组上皮细胞和上皮细胞的细胞间桥Fas/Apo - 1(CD95 )抗原阳性表达例数明显高于慢性成人牙周炎组和青少年牙周炎组 (P <0 .0 5 ) ;健康老年人组结缔组织中淋巴细胞和吞噬细胞Fas/Apo - 1(CD95 )抗原阳性表达例数明显低于其它 3组 (P <0 .0 5 )。结论 :由于感染性炎症和衰老的影响 ,①牙周炎患者和健康老年人牙龈组织中鳞状上皮凋亡细胞发生部位与身体其他器官鳞状上皮细胞凋亡发生部位不同 ;②在慢性老年牙周炎患者牙龈组织中上皮细胞和炎性细胞对细胞凋亡易感性     

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.     

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     

慢性牙周炎患者牙龈组织中IL-6、IL-34和M-CSFR的表达及临床意义

目的: 比较慢性牙周炎患者牙龈组织和健康牙龈组织中IL-6、IL-34及M-CSFR的表达水平,探讨IL-34 在慢性牙周炎发病中的作用。方法: 收集8例诊断为轻度慢性牙周炎患牙的牙龈组织和8例诊断为重度慢性牙周炎患牙的牙龈组织作为病例组,8例冠延长手术切除的牙龈组织作为对照组,应用实时荧光定量PCR检测IL-6、IL-34及M-CSFR mRNA的表达;应用蛋白免疫印迹检测IL-6、IL-34及M-CSFR蛋白的表达。使用GraphPad Prism 6.0对结果进行统计学分析。结果: IL-6、IL-34、M-CSFR mRNA及蛋白在慢性牙周炎牙龈组织中的表达水平均显著高于正常牙龈组织中的表达（P<0.05）。结论: IL-6、IL-34及M-CSFR的表达可能与慢性牙周炎密切相关。     

Apoptosis in chronic adult periodontitis analyzed by in situ DNA breaks, electron microscopy, and immunohistochemistry

BACKGROUND: Apoptosis is an evolutionary form of physiological cell death. Previous studies suggest that apoptosis is involved in the pathogenesis of periodontal diseases. Therefore, we studied the apoptotic events in the gingival tissue of chronic adult periodontitis patients. METHODS: Gingival tissue biopsies from 22 patients with chronic adult periodontitis and from 11 healthy controls were obtained. Criteria for patient inclusion in the periodontitis group were a minimum of 14 natural teeth, excluding third molars, with at least 10 posterior teeth; 5 to 6 sites with probing depth > or = 5 mm; attachment loss > or = 3 mm; and extensive radiographic bone loss. The control group included healthy subjects with no prior history of periodontal disease. Apoptosis was determined using the terminal TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique; electron microscopic analysis; and expression of Caspase-3, Fas, FasL, Bcl-2, and p53 by immunohistochemistry. RESULTS: TUNEL-positive cells and cells exhibiting chromatin condensation by electron microscopy were observed in the inflammatory infiltrate of biopsies obtained from periodontitis patients. Most of the TUNEL-positive cells belonged to neutrophil cell populations as they were stained with anti-myeloperoxidase. Positive staining for active-caspase 3, Fas, FasL, and p53 was only observed in the inflammatory infiltrate from periodontitis biopsies, whereas Bcl-2 cells were present in both periodontitis patients and healthy controls. CONCLUSIONS: Our findings establish that apoptosis is induced in the periodontal tissue by host and microbial factors and support the hypothesis that apoptotic mechanisms could be implicated in the inflammatory process associated with gingival tissue destruction observed in adult periodontitis patients.     

The expression of hBDs in the gingival tissue and keratinocytes from healthy subjects and periodontitis patients

ObjectiveAlthough the secretion of antimicrobial peptides in gingival tissue and isolated cells has been reported, the induction of human β-defensins (hBDs) in epithelial cells from the periodontitis patients was not stated before. This study aimed to compare the secretion of hBDs in gingival epithelial cells from periodontitis patients and healthy controls.DesignFirstly, gingival biopsies were obtained from chronic periodontitis patients and healthy controls and the hBDs expression level in gingival tissues was quantified. Then the epithelial cells from periodontitis patients and healthy controls were isolated and challenged with different concentrations of tumour necrosis factor-alpha (TNFα). The hBDs expression level was also quantified after induction. At last, to identify the molecular pathways involved in hBDs induction, the isolated cells were incubated with NF-kB or MAPK inhibitor before TNFα induction.ResultsHigher hBDs expression was found in gingival tissues from healthy controls. The in vitro experiments demonstrated that the hBD-2 expression in gingival epithelial cells from periodontitis patients can be induced by TNFα at lower dose, while the optimum expression level was much lower. The basal hBD-3 mRNA expression was much higher in cells from periodontitis patients. The molecular pathways involved in the responses to the inflammatory cytokine in patients and healthy controls were the same.ConclusionsThe epithelial cells from periodontitis patients are more prone to recognize and respond to TNFα to produce hBD-2. The basal expression of hBD-3 in keratinocytes from periodontitis patients suggested that hBD-3 may play an important role in the immunological reaction against periodontitis.     

牙周炎患者自身组织核酸内NLRP3 mRNA表达水平测定

目的:通过对牙周炎患者自身组织核酸内炎性小体复合物NLRP3,NLRC4及其相关基因IL-18,IL-1β,Caspase-1等mRNA表达水平的检测,明确炎性小体NLRP3在牙周炎中具有调控作用。方法:采集翻瓣术中慢性牙周炎患者炎性牙周组织以及正畸患者健康牙拔除术获取的健康牙周组织,提取总RNA逆转录cDNA ,-20 ℃冻存备用。Real-time PCR检测NLRP3、NLRC4、IL-18、IL-1β、Caspase-1 mRNA的表达。结果:与健康组牙周组织相比较,牙周炎患者自身组织核酸内NLRP3、IL-18、IL-1β、Caspase-1 mRNA的表达水平明显增高;而NLRC4 mRNA的表达在两组间差异不显著。结论:NLRP3及其相关基因IL-18、IL-1β、Caspase-1 mRNA在牙周炎患者自身组织核酸内的高表达,提示NLRP3在牙周炎的发病机制中具有影响作用。     

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。     

Apoptosis: an underlying factor for accelerated periodontal disease associated with diabetes in rats

Objectives

Diabetes mellitus (DM) is well-established risk factor for periodontal disease. DM can also lead to changes in the number of apoptotic cells in periodontal tissues. The goal of this study was to evaluate apoptosis, depending on DM, in healthy and diseased periodontal soft tissues.

Material and Methods

A total of 43 adult male Sprague–Dawley rats were used in this study. Experimental periodontitis was created by placing silk ligatures around the cervices of the first mandibular molars. Experimental diabetes was induced by intraperitoneal injection of the diabetogenic agent streptozotocin (STZ). Following the induction of both experimental diseases, the animals were divided into four groups: (1) The healthy group (H) (n?=?10); (2) The diabetes group (D) (n?=?10); (3) The periodontitis group (P) (n?=?11); and (4) The diabetes and periodontitis group (DP) (n?=?12). Apoptotic cells were determined by immunohistochemistry, and the frequency of apoptotic cells was evaluated by apoptotic index score.

Results

It was observed that there was less apoptosis in both the epithelial and gingival connective tissue cells of healthy diabetic tissues than in healthy tissues without diabetes. When periodontal disease existed, apoptosis increased in both the epithelial and gingival connective tissues of diabetic and non-diabetic animals.

Conclusions

There may be differences in the apoptotic mechanisms in the periodontal soft tissues of diabetic and non-diabetic animals.

Clinical relevance

Apoptosis may be one of the underlying factors in increased risk for periodontal disease that is associated with diabetes.     

Immunohistochemical localization of Toll‐like receptors 1–10 in periodontitis

Background/aim: In periodontitis, bacteria and pathogen‐associated molecular patterns are sensed by Toll‐like receptors (TLRs), which initiate intracellular signaling cascades that may lead to host inflammation. In this study, the expression and distribution of TLRs (TLR‐1 to TLR‐10) were immunohistochemically detected in gingival epithelium and connective tissue. Methods: Immunohistochemistry was used for the localization of TLRs in gingival tissue samples from 10 patients with chronic periodontitis and 10 healthy controls; these samples were obtained during routine periodontal flap operations and during extraction operations performed for retained wisdom teeth, respectively. For the evaluation, epithelial layers were stratified to basal, spinous, and superficial layers, and the percentages of TLR‐positive cells were determined. Results: Both healthy and periodontitis gingival tissues expressed all TLRs except TLR‐10. In patients with periodontitis, epithelial cells showed increased TLR expression towards the basal layer. Healthy controls showed more variable cellular TLR expression and distribution between the layers of epithelium. In the connective tissue, consistently higher TLR expression was found within the periodontitis group compared to the healthy group. Conclusions: For the first time, the cellular expression and distribution of TLR‐1 to TLR‐10 have been studied in periodontitis, indicating that TLR‐1 to TLR‐9 are differentially expressed both in connective tissue and epithelial layers. Except for TLR‐7 and TLR‐8, all the other TLRs showed statistically significant differences between patients with periodontitis and healthy controls, suggesting their involvement in the pathogenesis of periodontitis.     

Tumor necrosis factor-alpha expression and detection of apoptosis at the site of chronic periodontitis in AIDS patients

BACKGROUND: Apoptosis, or programmed cell death, is associated with the regulation of the life cell cycle of leukocytes in healthy and diseased states. OBJECTIVES: In the present study, we investigated the presence of apoptosis of mononuclear inflammatory cells in the periodontal lesion from adult periodontitis in healthy control patients and AIDS patients. MATERIALS AND METHODS: Tissue samples adjacent to a 5-6 mm gingival sulcus, measured with a periodontal probe, were obtained during routine periodontal surgical procedures. The direct immuno-peroxidase of digoxigenin-labeled genomic DNA method was used for in situ detection of apoptosis in gingival tissues. RESULTS: Many tumor necrosis factor-alpha (TNFalpha)-positive cells, detected by immunohistochemistry method, were observed in gingival samples of both groups of patients. In addition, a significant lower number ( p < 0.05) of mononuclear apoptotic cells were observed in AIDS patients when compared with healthy control patients. CONCLUSION: These data suggested an important role of the apoptosis of mononuclear cells in the pathogenesis of chronic adult periodontitis in AIDS patients.     

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.     

Collagenases in gingival crevicular fluid in type 1 diabetes mellitus

BACKGROUND: Studies have demonstrated that high levels of collagenase activity in gingival crevicular fluid (GCF) are associated with degradation of periodontal tissues in progressive periodontitis compared to periodontally healthy tissues. Because the activation of collagenases is an important issue in periodontitis, we have studied the activation of collagenase in gingival crevicular fluid samples of diabetic patients. METHODS: Collagenase activity was studied in human gingival crevicular fluids. Twenty-two poorly controlled diabetic patients (e.g., blood glucose: 11.0+/-0.7 mmol/l; hemoglobin A1c [HbA1c]: 9.6%+/-0.3%) and five well-controlled diabetic patients were compared to six chronic periodontitis subjects and five healthy controls. Collagenase activity against type I collagen was measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis quantitated by laser densitometry. RESULTS: The poorly controlled diabetic patients had more alveolar bone loss than the well-controlled diabetic subjects and controls (P<0.001; t test). The activity of collagenases in GCF in poorly controlled diabetic patients was similar to that seen in chronic periodontitis subjects (P>0.05) but higher than in healthy controls (P<0.01; t test), whereas there was no difference between the well-controlled diabetic subjects and systemically healthy controls (P>0.05; t test). CONCLUSION: Poorly controlled diabetes is strongly related to periodontal tissue destruction, and collagenases in GCF may mediate and reflect this effect.