增龄因素对鼠正畸牙齿移动中牙周组织骨保护素（OPG）表达的影响

目的 比较在正畸牙齿移动中幼年鼠和成年鼠牙周组织骨保护素(osteoprotegerin,OPG)信使RNA(mRNA)表达的不同,以探讨增龄因素影响正畸骨改建的分子机制。方法以4周(幼年鼠)和24周(成年鼠)雄性大鼠为实验动物,牵引左上第一磨牙近中移动为正畸牙齿移动模型,原位杂交检测牙周膜组织OPG mRNA的表达。结果 1.幼年鼠正畸牙齿移动中加力后3小时张力侧近牙槽骨表面可见OPG表达阳性细胞排列;与幼年鼠相比,成年鼠加力后牵张侧牙周膜细胞的OPG mRNA表达没有明显改变。2.幼年鼠加力后6小时开始压力侧即可观察到破骨细胞的数目明显增多,成年鼠加力后,压力侧加力24小时可见破骨细胞;无论幼年和成年鼠压力侧牙周膜细胞的OPG mRNA表达在加力前后均未见明显改变。结论 增龄因素使牙周组织内OPG表达明显增强;成年牙周组织中较强的OPG表达可能与成人正畸出现的牙槽骨吸收、牙齿移动迟缓相关。     

增龄因素对鼠正畸牙齿移动中牙周组织骨保护素(OPG)表达的影响

目的 :比较在正畸牙齿移动中幼年鼠和成年鼠牙周组织骨保护素 (Osteoprotegerin ,OPG)信使RNA(mRNA)表达的不同 ,以探讨增龄因素影响正畸骨改建的分子机制。材料与方法 :以 4周 (幼年鼠 )和 2 4周 (成年鼠 )雄性大鼠为实验动物 ,牵引左上第一磨牙中移动为正畸牙齿移动模型 ,右上第一磨牙为对照 ,利用OPG多相寡核苷酸探针原位杂交检测实验侧和对照侧牙周膜组织OPGmRNA的表达。结果 :1.幼年鼠对照侧近牙周膜的牙槽骨面可见破骨细胞和相应的骨陷窝 ;成年鼠牙周膜内细胞的OPGmRNA表达高于幼年鼠 ;2 .幼年鼠正畸牙齿移动中 ,加力后 6小时牵张侧近牙槽骨表面可见OPG表达阳性细胞排列 ,加力 2 4小时可见骨吸收陷窝内多核破骨细胞亦有明显OPGmRNA表达 ;压力侧加力 3小时开始即可观察到破骨细胞的数目明显增多 ,OPGmRNA在牙周膜细胞中的表达未见明显改变 ;3.与幼年鼠相比 ,成年鼠正畸牙齿移动中 ,牵张侧牙周膜细胞的OPGmRNA表达没有明显改变 ,压力侧加力 3小时仍未见典型的破骨细胞和骨吸收陷窝 ,加力 2 4小时见破骨细胞。结论 :增龄因素使牙周组织内OPG表达明显增强 ,加力后幼年牙周膜细胞较早表现出张力侧OPG表达增强和压力侧破骨细胞活动增强 ,幼年较成年的牙周组织有较强的骨改建能力。     

增龄因素对鼠正畸牙牙周组织骨保护素及其配体表达的影响

目的:比较幼年鼠和成年鼠在正畸牙移动中牙周组织骨保护素(OPG)及其配体(OPGL)表达的不同比值,探讨增龄因素对正畸骨改建影响的分子机制。方法:制备大鼠正畸牙齿移动模型,于牙齿移动1d、3d、5d、7d、10d、14d及14d后去除正畸力1周后取材,免疫组化检测牙周组织OPG和OPGL的表达。结果:①.牙齿移动3d时,幼年鼠压力侧OPGL的表达明显增强;而成年鼠此时OPGL的表达没有幼年鼠明显。②.牙齿移动5d和7d时,幼年鼠和成年鼠OPGL的表达均成强阳性,破骨细胞多。③.牙齿移动10d、14d时,幼年鼠和成年鼠OPGL的表达逐渐减弱。④.14d时去除正畸力至21d时发现幼年鼠和成年鼠OPGL均已成弱阳性表达,幼年鼠可见部分成骨细胞。结论:在正畸力的作用下,OPGL与OPG的表达比值与年龄关系密切,增龄因素引起的牙周组织内OPGL/OPG表达变化可能是导致成年正畸特点的分子机制之一。     

体外培养的人牙周膜细胞在机械力作用下骨保护因子mRNA的表达

目的研究机械力对体外培养的人牙周膜细胞骨保护因子(osteoprotegerin,OPG)信使RNA(messenger RNA,mRNA)表达的影响.方法应用细胞加力装置对体外培养的人牙周膜细胞施予间歇性牵张力48小时,原位杂交检测细胞加力前后OPG mRNA的表达.结果机械力作用下人牙周膜细胞OPG mRNA表达下降.结论正畸骨改建过程中,机械力引起牙周膜细胞OPG表达的变化.这一结论为进一步研究人牙周膜细胞在机械力作用下参与骨改建的机理打下基础.     

机械力对人牙周膜细胞分化的影响

目的 :正畸治疗的一个最基本现象就是牙齿受力后牙周组织发生改建 ,从而发生牙齿移动。但是机械力如何转化为牙周组织的生物学变化至今仍不十分清楚。牙周膜细胞是机械力作用的靶细胞 ,具有多种分化潜能 ,因此我们在离体状态观察机械力作用下人牙周膜细胞的表型及与骨代谢密切相关的功能蛋白骨保护素 (osteoprotegerin ,OPG)表达的变化 ,以期进一步了解正畸过程中牙槽骨改建的生物学机理。方法 :组织块法培养人牙周膜细胞 ,应用自行研制的体外细胞加力装置 ,对细胞施予间歇性牵张力 ,加力 2 4小时 ,分别在加力后 0 ,2 ,4 ,6 ,12 ,2 4小时 ,用生化法检测牙周膜细胞碱性磷酸酶 (alkalinephosphotase,ALP)的表达 ,放射免疫法检测骨钙素 (osteocalcin ,OCN)的表达 ,通过原位杂交和酶联免疫吸附实验 (Enzyme linkedimmunoabsordentassay ,ELISA)从mRNA和蛋白水平检测人牙周膜细胞OPG表达的变化。结果 :机械力作用下 ,在 2 4小时加力周期内 ,间歇性牵张力作用增强人牙周膜细胞ALP和OCN蛋白表型的表达。而与破骨细胞分化关系十分密切的功能蛋白OPG表达降低。在开始受力的数小时 ,人牙周膜细胞OPG表达即发生变化 ,且变化量明显 ;在受力 12小时至 2 4小时期间 ,OPG表达降低量不如早期明显。结论 :机械力可诱导人     

低能量激光对正畸力作用下牙周膜细胞骨改建相关因子mRNA表达的影响

目的 研究低能量激光对正畸张应力及压应力作用下人牙周膜细胞骨改建相关因子mRNA表达的影响.方法 培养人牙周膜细胞,模拟正畸加力,结合低能量激光照射,在不同条件下(激光、应力、激光+应力)培养6 h,通过Realtime PCR检测IL-1β、Runx2、OPN、OPG、RANKL mRNA的表达并计算RANKL/OPG的比值.结果 人牙周膜细胞在培养6 h后,IL-1β、Runx2、OPN、OPG、RANKL mRNA的表达都呈现出激光+应力组大于单纯应力组及单纯激光组.结论 模拟正畸加力及低能量激光照射均可使得人牙周膜细胞骨改建相关因子的表达量增加,两者可起到协同作用,促进正畸牙齿移动过程中的牙槽骨改建.     

白细胞介素-6与牙周组织的改建

白细胞介素-6是一种来源广泛的多功能细胞因子,与牙周组织改建有密切的关系。咬合力废用和代偿,均诱导牙周膜细胞和牙槽骨成骨细胞表达IL-6蛋白及mRNA明显增加。IL-6通过作用于破骨细胞前体来促进骨吸收,也能够抑制牙周膜细胞的生长,从而影响组织的修复和代谢功能;它还能促进成骨细胞的分化,促进骨的形成,是牙周组织改建的重要细胞因子。     

壳寡糖对牙周炎大鼠牙槽骨吸收及Th17/Treg平衡和OPG/RANKL/RANK通路的影响

目的: 探讨壳寡糖对牙周炎大鼠牙槽骨吸收及Th17/Treg平衡和OPG/RANKL/RANK通路的影响。方法: 建立牙周炎大鼠模型,随机分为模型组、壳寡糖低剂量组、壳寡糖中剂量组、壳寡糖高剂量组和甲硝唑组,每组12只,另取12只作为对照组。分组处理后,评估牙龈指数、牙槽骨吸收值;H-E染色观察牙周组织病理形态学变化;流式细胞术检测外周血中Th17/Treg细胞比值;酶联免疫吸附试验（ELISA）检测各组大鼠血清中IL-17、TGF-β、RANKL、OPG水平,实时荧光定量PCR（qRT-PCR）检测各组大鼠牙周组织OPG、RANKL mRNA表达水平。采用SPSS 24.0软件包对数据进行统计学分析。结果: 与对照组相比,模型组大鼠牙周组织呈现牙周膜纤维束断裂、排列紊乱,毛细血管扩张、增生,炎症细胞浸润等病理损伤;牙龈指数、牙槽骨吸收值、Th17/Treg比值、血清RANKL及IL-17水平、牙周组织RANKL mRNA水平显著升高（P<0.05）,血清OPG及TGF-β水平、牙周组织OPG mRNA水平显著降低（P<0.05）。与模型组相比,壳寡糖低、中、高剂量组和甲硝唑组大鼠牙周组织病理损伤减轻;牙龈指数、牙槽骨吸收值、Th17/Treg比值、血清RANKL及IL-17水平、牙周组织RANKL mRNA水平显著降低（P<0.05）,血清OPG及TGF-β水平、牙周组织OPG mRNA水平显著升高（P<0.05）,且壳寡糖各组呈剂量依赖性,壳寡糖高剂量组与甲硝唑组相比,差异无统计学意义（P>0.05）。结论: 壳寡糖可促使Th17/Treg平衡恢复正常,上调OPG表达,下调RANKL表达,抑制牙周炎大鼠牙槽骨吸收,改善其临床症状。     

慢性牙周炎中核因子κB受体活化子、护骨素和肿瘤坏死因子-α的蛋白表达

目的　比较慢性牙周炎牙槽骨吸收处肉芽组织中的核因子κB受体活化子 (RANKL)、护骨素 (OPG)和肿瘤坏死因子(TNF) -α的水平 ,研究慢性牙周炎牙槽骨吸收与这些调节因子间的关系。方法　应用鼠抗人单克隆抗体、半定量分析及数字成像技术分析慢性牙周炎组织中RANKL、OPG和TNF α的蛋白表达。结果　慢性牙周炎牙槽骨吸收处肉芽组织中有较高的RANKL和TNF α蛋白表达 ,OPG蛋白表达相对较低 (P <0 .0 5 ) ;非牙周炎组织中显示较强的OPG蛋白表达和较弱的RANKL和TNF -α蛋白水平 (P <0 .0 5 ) ;OPG蛋白表达与牙周组织中内皮细胞相关。结论　RANKL可能与慢性牙周炎牙槽骨吸收相关 ;RANKL和OPG平衡的变化在慢性牙周炎骨吸收的分子机制中起重要调节作用 ;TNF α可能协同参与牙槽骨的吸收     

Cytokine pattern determines the progression of experimental periodontal disease induced by Actinobacillus actinomycetemcomitans through the modulation of MMPs, RANKL, and their physiological inhibitors

OBJECTIVE: Inflammatory and immune reactions raised in response to periodontopathogens are thought to trigger periodontal tissue destruction. We therefore investigated the expression of matrix metalloproteinases (MMPs) and the osteoclastogenic factor RANKL (receptor activator of nuclear factor-kappaB ligand), their respective inhibitors TIMPs (tissue inhibitors of metalloproteinases) and OPG (osteoprotegerin) and their possible correlation with the expression of inflammatory and regulatory cytokines in the course of experimental periodontal disease in mice. METHODS: We characterized the time course of leukocyte migration and alveolar bone loss in C57BL/6 mice infected with Actinobacillus actinomycetemcomitans. Quantitative polymerase chain reaction (RealTime PCR) and ELISA were performed to determine the expression of MMPs, TIMPs, RANKL, OPG and cathepsin K, interleukin-1beta, tumor necrosis factor-alpha, interferon-gamma, interleukin-12, interleukin-4 and interleukin-10 in periodontal tissue samples harvested throughout the course of experimental disease. RESULTS: Oral inoculation of A. actinomycetemcomitans results in an intense and widespread migration of leukocytes to the gingival tissues, besides marked alveolar bone resorption. Our data also demonstrate two distinct patterns of MMP/TIMP and RANKL/OPG expression in the course of experimental periodontal disease. The expression of MMPs (MMP-1, 2 and 9) and RANKL was correlated with the expression of interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma, in a time period characterized by the intense increase of inflammatory reaction and alveolar bone loss. On the other hand, interleukin-4 and interleukin-10 were associated with higher expression of TIMPs (TIMP 1, 2 and 3) and OPG, with a lower expression of MMPs and RANKL, and with reduced rates of increase of cellular infiltration in periodontal tissues and alveolar bone loss. CONCLUSIONS: It is possible that the pattern of cytokines produced in periodontal tissues determines the progression and the severity of experimental periodontal disease, controlling the breakdown of soft and bone tissues through the balance between MMPs/TIMP and RANKL/OPG expression in gingival tissues.     

Compensatory bone formation in young and old rats during tooth movement

The aim of this study was to investigate compensatory lingual alveolar bone formation during tooth movement in young and old rats, using the vital bone marker tetracycline. Wistar male rats were separated into the following groups: 13-week-old rats without appliances (13C: control, n = 5), 60-week-old rats without appliances (60C: control, n = 5), 13-week-old rats with appliances (13E: experimental, n = 10), and 60-week-old rats with appliances (60E: experimental, n = 10). The upper first molars of the 13E and 60E groups were moved lingually using fixed appliances. On the third day of tooth movement, tetracycline (TC) was intra-peritoneally injected in all animals including the controls. On the 21st day of tooth movement, the animals were killed and unfixed, and undecalcified, 5-microm frozen frontal sections of the rat first molar areas in both control and experimental groups were examined under light and fluorescent microscopes. In the 13C group without tooth movement, tetracycline labelling lines were obvious in the alveolar crest, apical areas, and interradicular septum, indicating vertical alveolar bone growth. However, in the 60C control group, tetracycline labelling was almost undetectable throughout the alveolar bone. Although the lingual alveolar crest was resorbed from the periodontal side after lingual tooth movement, the sharp, bright labelling lines were still present from the crest to the lingual periosteal alveolar bone in the 13E group. In the 60E group the lines appeared in the lingual periosteal alveolar bone containing the crest, indicating considerable new bone formation. The results indicate that compensatory bone formation occurs in the alveolar crest area and, consequently, alveolar bone height is maintained, even in aged rats.     

老龄鼠实验性牙周炎的免疫特点

目的： 通过口腔涂抹牙周致病菌的方法建立小鼠牙周炎模型,比较低龄小鼠与老龄小鼠牙周炎发病免疫状态差异,分析Th17/Treg细胞相关因子在不同年龄牙周炎小鼠中的表达情况。方法：采用实体显微镜评价牙周骨吸收的程度,H-E染色法观察牙周组织病变的炎细胞浸润情况,TRAP染色法观察牙周骨组织表面破骨细胞浸润情况,实时定量PCR检测牙龈组织中Th17/Treg细胞相关因子TGF-β1、IL-10、IL-17A、RANKL mRNA的表达水平。采用SPSS 17.0软件包对数据进行统计学分析。结果：实验4周后,牙周炎组与正常对照组相比,釉-牙骨质界至牙槽嵴顶的距离显著增加（P<0.01）,老龄组小鼠釉-牙骨质界至牙槽嵴顶的距离比低龄组小鼠显著增加（P<0.01）;牙周组织中大量炎细胞浸润,牙周袋加深;牙周组织中 TGF-β1、IL-10 mRNA的表达水平显著降低（P<0.01）,IL-17A、RANKL mRNA表达水平显著增高（P<0.01）。结论：老龄小鼠牙周炎中炎症介质的表达存在异常状态,比低龄小鼠牙周组织病损状态加剧,提示牙周炎的炎症状态可能不仅与其分泌的免疫抑制性细胞因子表达降低有关,更与其增龄性相关。     

Local osteoprotegerin gene transfer to periodontal tissue inhibits lipopolysaccharide-induced alveolar bone resorption

Background and Objective:  Osteoclastogenesis is primarily activated by receptor activator of nuclear factor κB ligand (RANKL) and is inhibited by osteoprotegerin (OPG). A previous study demonstrated that local OPG gene transfer to periodontal tissue inhibited RANKL-mediated osteoclastogenesis and experimental tooth movement. In the present study, we tested the hypothesis that local OPG gene transfer to the periodontium can neutralize RANKL activity induced by lipopolysaccharide injection, thereby inhibiting osteoclastogenesis and diminishing alveolar bone resorption in experimental periodontal disease.
Material and methods:  Seven-week-old male Wistar rats received an injection of lipopolysaccharide or phosphate-buffered saline in the palatal gingiva of the upper first molars on both the right and left sides. An inactivated haemagglutinating virus of Japan (HVJ) envelope vector containing a mouse OPG expression plasmid [pcDNA3.1(+)-mOPG] or mock vector was injected periodically into the palatal periodontal tissue of the upper first molars.
Results:  Lipopolysaccharide injection induced severe periodontal bone resorption. Local OPG gene transfer induced OPG production, and osteoclastogenesis was inhibited. Local OPG gene transfer significantly decreased alveolar bone resorption.
Conclusion:  Osteoprotegerin gene transfer to periodontal tissue inhibited osteoclastogenesis and alveolar bone resorption in lipopolysaccharide-induced experimental periodontal disease.     

糖尿病大鼠牙周组织中免疫调节相关信号通路研究

目的观察糖尿病大鼠模型牙周组织中CD4、CD8、核因子-κB受体活化子配体(receptor activator forNF-κB ligand,RANKL)及骨保护素(osteoprotegerin,OPG)表达的变化。方法 38只健康雄性SD大鼠,随机选取30只,高热量饲料喂养1个月后,建立肥胖模型,随后每只大鼠按55 mg/kg一次性静脉注射链脲佐菌素(strepto-zotocin,STZ),建立糖尿病模型组;其余8只普通饲料喂养1个月后,一次性静脉注射相同剂量的pH值4.5、0.1 mol/L柠檬酸缓冲液,设为对照组。建模第16周处死大鼠,解剖上颌骨,制作3μm厚连续切片,荧光免疫组化检测牙周组织中CD4、CD8及RANKL、OPG的表达。采用激光扫描共聚焦显微镜(laser scanning confocal micro-scope,LSCM)采集图像,行灰度值分析,比较CD4、CD8、RANKL、OPG在糖尿病组和对照组大鼠牙周组织中的表达差异。结果糖尿病组CD4、CD8、RANKL的表达均高于对照组,差异有统计学意义(P<0.05);糖尿病组与对照组相比OPG表达降低,差异无统计学意义。结论糖尿病可能通过RANKL/OPG调节途径促进牙周炎的发生发展。