非受体型蛋白酪氨酸磷酸酶2与核因子-κB在糖尿病牙周炎牙周组织破坏中的作用研究

目的观察糖尿病牙周炎条件下小鼠牙周组织中非受体型蛋白酪氨酸磷酸酶2（PTPN2）、核因子-κB（NF-κB）的表达与牙周组织破坏的关系。方法将4周龄健康雄性C57BL/6J小鼠随机分为正常对照组（N组）、单纯牙周炎组（P组）及糖尿病牙周炎组（DP组）,每组6只。采用口内接种牙龈卟啉单胞菌法在P组建立牙周炎模型,采用腹腔注射链脲佐菌素结合高脂高糖食物与口内接种牙龈卟啉单胞菌法在DP组建立糖尿病牙周炎模型。各组动物于P、DP组末次细菌接种4周后处死,通过体视显微镜观察牙槽骨形态和附着丧失面积,采用苏木精-伊红（HE）染色法观察牙周附着丧失的高度,免疫组织化学法检测牙周组织中PTPN2与NF-κB的表达强度。结果P、DP组牙槽骨的附着丧失面积和牙周附着丧失高度均高于N组（P<0.05）,PTPN2的表达量低于N组（P<0.05）,而NF-κB的表达量则高于N组（P<0.01）。结论在糖尿病牙周炎的发生发展过程中,NF-κB可能有促进作用,而PTPN2可能有抑制作用;PTPN2的表达减少可能导致NF-κB表达增强而加重牙周组织破坏。     

大鼠偏侧咀嚼牙槽骨及牙周组织环氧合酶-2分子的表达和分布

目的：观察环氧合酶-2（cyclooxygenase-2,COX-2）在大鼠偏侧咀嚼牙槽骨及牙周组织中的表达和分布特点,探讨其在牙槽骨吸收、新生和重建中的作用机制。方法：建立大鼠偏侧咀嚼模型,制备不同时期大鼠咀嚼侧牙槽骨标本,行兔抗大鼠COX-2多克隆抗体（1：200）免疫组织化学染色。结果：在正常组牙周膜周围固有牙槽骨中散在血管内皮细胞胞浆呈弱阳性着色：3d实验组咀嚼侧见部分牙槽骨成骨细胞及散在牙周膜细胞胞浆呈较强阳性着色：1w实验组咀嚼侧为阴性着色;2w和4w实验组咀嚼侧见部分牙周膜细胞胞浆局部呈强阳性着色。结论：偏侧咀嚼改变了牙槽骨及牙周组织所处的生物力学环境,进而诱导了体内牙槽骨成骨细胞和牙周膜细胞COX-2的表达。     

细胞外基质磷酸糖蛋白在大鼠牙周组织缺损愈合过程中的表达

目的:观察细胞外基质磷酸糖蛋白(matrix extracellular phosphoglycoprotein,MEPE)在大鼠牙周组织缺损愈合过程中的表达,初步探讨MEPE在牙周组织再生中可能发生的作用。方法:24只成年健康雄性Wistar大鼠随机分2组:术后14 d组和术后28 d组各12只,行左侧下颌骨开窗术,制备牙周组织缺损模型,HE染色观察牙周组织缺损愈合情况,免疫组织化学染色法观察MEPE在牙周组织缺损愈合过程中各组织的表达。结果:术后14 d,术区有部分新生牙槽骨形成,部分牙周纤维附着于根面牙本质上;术后28 d,术区完全被新生牙槽骨覆盖,在新生牙槽骨和牙本质之间可见牙周膜和部分牙骨质生成,牙周膜大部分附着于根面上;牙周组织缺损愈合过程中,MEPE在健康对照侧仅呈弱阳性表达,而在缺损区牙槽骨和牙周膜相关细胞中均呈阳性表达。结论:在牙周组织缺损愈合过程中,细胞外基质磷酸糖蛋白参与牙槽骨以及牙周膜的再生。     

大鼠磨牙及牙周组织中神经纤维分布:神经丝蛋白免疫组织化学研究

目的：观察大鼠磨牙及牙周组织神经纤维分布特点。方法：大鼠磨牙及其牙槽骨脱钙后切征并行神经丝蛋白（ＮＦＰ）免疫,组织化学染色。结果：大鼠磨牙、牙周膜及牙龈中神经纤维丰富,神经丝蛋白阳性神经纤维在髓角处形成牙本质细胞下丛,其中一些神经纤维进入牙本质。在牙周膜,神经丝蛋白阳性神经纤维在牙根下段密集分布 。因牙周膜内存在两型神经来梢：游离神经来梢及Ｒｕｆｆｉｎｉ终末。前者在整个牙周膜内皆有分布。后者常成组     

富含亮氨酸的间质蛋白多糖在小鼠牙周组织发育中的组织学定位

目的研究富含亮氨酸的蛋白多糖类物质：纤维调节素（FMN）、核心蛋白聚糖（DCN）及双糖链蛋白聚糖（BGN）在小鼠牙周组织不同发育时期的分布特点，探讨其在牙周组织发育中的作用。方法取出生后第5、10、15、25天的BALB／c小鼠36只，引颈处死，解剖并分离含下颌第一磨牙区的下颌骨，新鲜配制4％多聚甲醛固定24h，甲酸一甲酸钠复合脱钙液脱钙1～3周，常规石蜡包埋，近远中向5μm连续切片。免疫组织化学Power Vision^TM二步法，观察各发育时期第一磨牙牙周组织中3种间质蛋白多糖的组织学分布特点。结果FMN在牙龈结缔组织和牙周膜中呈强阳性表达，且在牙周膜与牙骨质及牙槽骨界面有较强表达；DCN强阳性表达于牙龈结缔组织、靠近牙槽骨面的牙周膜及牙槽骨表面的成骨细胞中；BGN则在各期牙龈结缔组织及牙周膜中表达，在牙槽骨表面及成骨细胞中为阴性。结论FMN可能与DCN及BGN相互作用，调节牙龈结缔组织及牙周膜胶原纤维的网络形成，并可能参与牙槽骨和牙骨质的矿化过程。     

咬合力增强对老年大鼠磨牙牙周组织成骨和增殖细胞核抗原表达的影响

目的研究咬合力增强对老年大鼠牙周组织成骨和增殖细胞核抗原(PCNA)表达的影响。方法建立咬合力增强动物模型,运用伊红(HE)染色、碱性磷酸酶(ALP)染色、抗酒石酸酸性磷酸酶(TRAP)染色和PCNA免疫组织化学染色检测牙周组织成骨和PCNA表达的变化。结果咬合力增强2周时牙周膜结构致密,牙槽骨骨壁凹凸不平,同时可见交替出现的骨形成区;4周时牙周膜细胞PCNA阳性率最高,6周时牙槽骨表面成骨细胞的ALP染色强阳性,成骨活跃;而各组破骨细胞数目无显著差异。结论老年大鼠的牙周支持组织能在一定范围内耐受高于生理状态的咬合力,为临床上老年患者制作固定义齿提供了理论依据。     

糖尿病大鼠牙周组织改变的实验观察

目的 探索大鼠发生糖尿病后对其牙周组织的影响.方法 建立糖尿病大鼠动物模型,分别在1、3和6个月三个不同时期,用光镜观察和免疫组织化学染色方法研究大鼠的牙周组织改变.结果 糖尿病大鼠1个月时即有牙槽骨的吸收破坏, 3个月至6个月时骨吸收范围扩大,牙周纤维排列紊乱和断裂.骨吸收仅位于牙槽骨侧,而牙体侧牙骨质和牙周膜无破坏.结论 大鼠在发生糖尿病后,出现特异性牙槽骨吸收为主要特征的病理性改变.     

大鼠牙周组织中肿瘤坏死因子—α受咬合力产强影响变化的研究

目的：S倘交合力在正常及增强状态下,大鼠牙周细胞TNF－α动态表达,探讨TNF－α在牙周组织织改建中的分子机理,方法：采用HE染色和免疫组化的方法,观察牙周形态变化以及牙周组织中TNF－α蛋白表达,结果：咬合力增强引起牙周膜增宽、牙槽骨形成。牙周细胞TNF－α表达较正常咬合力时明显增强。结论：咬合力增强,促使牙周组织产生TNF－α明显增多,诱发了破骨功能,同时,还激活了成骨功能,本实验从子水平探讨了牙周组织改建的机理;揭示了牙周组织结构与功能在改中的一致性。     

胰岛素治疗对糖尿病大鼠牙槽骨RANKL/OPG mRNA影响的研究

目的通过研究胰岛素治疗对糖尿病大鼠牙周组织病理改变及牙槽骨中NF-κB受体活化因子配体(Receptor activator of nuclear factor-κB ligand,RANKL)和骨保护素(osteoprotegerin,OPG)mRNA水平比值情况的影响,探讨糖尿病影响牙周病时牙槽骨吸收的机理。方法将12只大鼠采用静脉注射链脲佐菌素的方法建立糖尿病模型,并随机分为治疗组和对照组。治疗组给予胰岛素皮下注射,对照组注射等量生理盐水。分别于实验开始时、造模成功后和8周后处死时测量大鼠体重和血糖。右下磨牙区牙周组织脱钙后HE染色观察组织病变状况;应用RT-PCR检测左下磨牙区牙槽骨RANKL和OPG mRNA表达情况,并比较两组大鼠RANKL/OPG比值差异。结果胰岛素治疗组较糖尿病组牙周组织炎症反应减轻,牙槽骨吸收减弱;血糖值(P<0.05)及RANKL/OPGmRNA比值(P<0.01)降低。结论胰岛素治疗可能增加牙周组织修复和再生能力,降低糖尿病大鼠的牙槽骨RANKL/OPGmRNA比值。提示血糖水平增高可能是影响糖尿病大鼠的牙槽骨吸收危险因素之一。     

力增强影响大鼠牙周组织前列腺素(PGE2)表达变化的研究

目的　检测力在正常及增强状态下 ,大鼠牙周细胞 PGE2 的动态表达 ,初探 PGE2 在牙周组织改建中的分子机理。方法　采用 HE染色和免疫组化的方法 ,观察牙周形态变化以及牙周组织中 PGE2 蛋白表达。结果　力增强引起牙周膜增宽、牙槽骨形成。牙周细胞中 PGE2 表达较正常力时明显增强。结论　力增强 ,促使牙周组织产生 PGE2 明显增多 ,诱发了破骨功能 ;同时 ,还激活了成骨功能。本实验从分子水平探讨了牙周组织改建的机理。     

Vitamin C intake inhibits serum lipid peroxidation and osteoclast differentiation on alveolar bone in rats fed on a high-cholesterol diet

Objective

A high-cholesterol diet stimulates osteoclast differentiation, which may be induced by increased serum lipid peroxidation. The inhibition of serum lipid peroxidation by vitamin C may offer beneficial effects on osteoclast differentiation including increased expression of receptor activator of nuclear factor (NF)-κB ligand (RANKL) and NF-κB. This study investigated the effects of vitamin C intake on RANKL and NF-κB expression in periodontal tissue of rats fed a high-cholesterol diet.

Design

Twenty-four rats (8 weeks old) were divided into four groups: a control group (fed a regular diet) and three experimental groups (fed a high-cholesterol diet supplemented with 0, 1 and 2 g/l vitamin C/day) in this 12-week study. Vitamin C was provided by its addition to drinking water. As an index of serum lipid peroxidation, hexanoyl-lysine (HEL) level was determined by a competitive enzyme-linked immunosorbent assay method. Immunohistological analysis was performed to evaluate RANKL and NF-κB expression on the alveolar bone surface. The number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was also counted.

Results

Feeding a high-cholesterol diet increased not only the serum HEL level but also the number of TRAP-positive osteoclasts on the alveolar bone surface, with an increase in RANKL and NF-κB expression on alveolar bone surface. Intake of vitamin C reduced the serum HEL level and osteoclast differentiation, with decreasing RANKL and NF-κB expression.

Conclusions

Vitamin C intake could suppress osteoclast differentiation, including RANKL and NF-κB expression on the alveolar bone surface, by decreasing serum lipid peroxidation in rats fed a high-cholesterol diet.     

HDAC5-Mediated Acetylation of p100 Suppresses Its Processing

IntroductionPeriodontitis is a condition involving chronic inflammation in the gums, periodontal ligaments, cementum, and alveolar bone. Nuclear factor-κB (NF-κB) activation is the prominent mediator of inflammation and osteoclast differentiation. The role of histone deacetylase 5 (HDAC5) in periodontitis development and NF-κB regulation is not fully understood.MethodsWe used primary mouse bone marrow–derived osteoclast cultures in vitro and a mouse model of chronic periodontists (CPD) treated with the HDAC4/5 inhibitor LMK-235. Real-time polymerase chain reaction, micro computed tomography, flow cytometry, western blot, and immunoprecipitation were used to study proinflammatory cytokines, NF-κB activation, HDAC5 activity, and the interaction of HDAC5 with NF-κB p100.ResultsLMK-235, a selective inhibitor of HDAC4 and HDAC5, reduced osteoclast marker gene expression (Cstk, Acp5, and Calcr) and tartrate-resistant acid phosphatase activity in primary osteoclast cultures. LMK-235 reduced the increase in cementoenamel junction–alveolar bone crest distance, inflammatory cell infiltration of gingival tissues, and expression levels of interleukin (IL)-1β, tumor necrosis factor alpha, IL-6, and IL-23a, indicating an ameliorative effect on CPD. Immunoprecipitation experiments have further confirmed p100–HDAC5 interaction, acetylation levels of p100, and NF-κB activation.ConclusionsThese results indicate that HDAC5 binds and deacetylates p100, leading to its activation, increased proinflammatory cytokine production, gingival infiltration, and osteoclast differentiation, thus promoting alveolar bone resorption. HDAC5 inhibition is therefore a potentially promising therapeutic strategy for the treatment of periodontitis.     

Immunohistochemical localisation of extracellular matrix proteins in the periodontium during cementogenesis in the rat molar

OBJECTIVE: The development of the periodontium involves the coordinated expression of numerous extracellular matrix (ECM) macromolecules and their receptors (integrins). The aim of this study was to determine the expression of selected hard and soft tissue matrix molecules and the integrin alpha5beta1 in the periodontal tissues, during cementogenesis in the rat molar. METHODS: Using immunohistochemical methods, the distribution of the extracellular matrix proteins, fibronectin, tenascin, and bone sialoprotein (BSP), as well as the integrin subunits alpha5 and beta1 were studied in rats aged 3, 5 and 8 weeks. RESULTS: Fibronectin was widely distributed in the gingival epithelium, gingival connective tissue and in the periodontal ligament. Tenascin expression was less marked compared with fibronectin, but was more distinctly associated with cells and peri-cellular areas of the epithelial-connective tissue interface, the gingiva and within the periodontal ligament. The fibronectin-receptor alpha5beta1 integrins were expressed by epithelial cells, periodontal ligament cells and gingival fibroblasts. A notable finding was the increased staining intensity of fibronectin, tenascin and alpha5beta1 integrin in all 5-week old molar sections in the periodontal ligament matrix and cells, apical to the cemento-enamel junction (CEJ) along the alveolar crest (AC) ridge height. Bone sialoprotein was distinctly associated with the hard tissues of the periodontium as acellular cementum and alveolar bone matrix expressed bone sialoprotein throughout all sections, in all age groups. CONCLUSIONS: In conclusion, this study has demonstrated the selective distribution of several hard and soft tissue matrix molecules during periodontogenesis. The results highlight the complex nature of interactions of various proteins and molecules during development. The interactions between these molecules and their specific roles in development and regeneration await further investigation.     

A single application of hydrogen sulphide induces a transient osteoclast differentiation with RANKL expression in the rat model

Objective

Oral malodor is mainly attributed to volatile sulphur compounds (VSCs) such as hydrogen sulphide (H₂S), methyl mercaptan and dimethyl sulphide. VSC accelerate periodontal soft tissue destruction. However, there is little information about the potential role of H₂S in alveolar bone loss. The purpose of this animal study was to examine the effects of sodium hydrogen sulphide (NaHS), H₂S donor drug, on osteoclast differentiation in rat periodontal tissue.

Design

Twenty-four male Wistar rats (8 weeks old) were divided into four groups: a control group and three experimental groups, which were examined at 3 h, 1 day, and 3 days after topical application of 3 μl NaHS (l M in physiological saline) into the gingival sulcus of rat first molar. Expression of tumour necrosis factor (TNF)-α, RANKL, NF-κB and tartrate-resistant acid phosphatase (TRAP) was evaluated in the periodontal tissue.

Results

Three hours after NaHS application, TNF-α expression increased in the periodontal ligament. The numbers of RANKL-positive osteoblasts and TRAP-positive osteoclasts significantly increased progressively with time and reached a maximum level after 1 day. Significant up-regulation of RANKL and NF-κB mRNA was observed at 3 h after NaHS application.

Conclusions

H₂S application caused a transient increase of osteoclast differentiation with up-regulation of RANKL expression in osteoblasts. H₂S, which is primarily responsible for halitosis, may also contribute to alveolar bone resorption through RANKL expression.     

白藜芦醇减轻LPS诱导的牙周膜细胞损伤并抑制TLR4/NF-κB活化

目的:探究白藜芦醇(RES)对脂多糖(LPS)诱导的人牙周膜细胞(hPDLCs)损伤的保护作用.方法:体外培养并鉴定hPDLCs,将培养的hPDLCs随机分为5组:对照组、LPS(10 μg/ml)+RES(0、30、60、90tμmol/L)组,MTT法检测各组细胞增殖能力,ELISA检测各组细胞分泌TNF-α/IL-6水平,PCR和Western blot检测各组细胞TLR4/NF-κB mRNA和蛋白表达.结果:分离培养的hPDLCs抗波丝蛋白表达阳性,抗角蛋白表达阴性.与对照组比,LPS处理后细胞增殖能力明显降低,细胞分泌TNF-α/IL-6水平和TLR4/NF-κB mRNA和蛋白表达明显增加;30～90 μmol/L白藜芦醇预处理后,细胞增殖能力增加(P＜0.05),细胞分泌TNF-α/IL-6水平、TLR4/NF-κB mRNA以及蛋白表达则下调(P＜0.05),均呈现一定的浓度依赖性.结论:白藜芦醇可抑制TLR4/NF-κB活化并减轻LPS诱导的牙周膜细胞损伤.     

PPARγ在脂多糖刺激牙周膜细胞中调控NF-κB信号通路的作用研究

目的:探讨PPARγ在牙周炎中调控作用机制。方法:组织块法培养健康人牙周膜细胞(human periodontal ligament cells, hPDLCs)并鉴定。细胞处理分为以下4组,A组:对照组;B组:脂多糖(lipopolysaccharide,LPS )刺激组;C组:罗格列酮对照组,即二甲基亚砜(dimethylsulfoxide, DMSO)处理组;D组:罗格列酮处理组。免疫印迹法检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptor γ,PPARγ)和核因子κB(nuclear factor κB, NF-κB) p65胞核、胞浆及总蛋白含量,细胞免疫荧光检测NF-κB p65表达部位。实时定量PCR和酶联免疫法检测白细胞介素1β(interleukin-1β,IL-1β)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)RNA和蛋白表达。结果:LPS刺激下,胞核PPARγ表达降低NF-κB p65表达升高,相应的IL-1β和TNF-α RNA及蛋白表达量均升高。同时加入罗格列酮后,胞核PPARγ表达升高NF-κB p65表达降低,且IL-1β和TNF-α RNA及蛋白表达量均降低。差异均有统计学意义(P<0.01)。结论:PPARγ通过下调NF-κB信号通路抑制脂多糖刺激下牙周膜细胞炎症因子RNA表达和蛋白分泌,进而调节牙周炎症反应。