P物质受体在体外培养破骨细胞内的表达及意义

目的观察P物质受体(NK1)在体外培养大鼠破骨细胞内的表达情况。方法采用细胞培养和免疫组化ABC方法,检测NK1受体在大鼠破骨细胞内的表达。结果NK1受体在大鼠破骨细胞内均呈阳性表达,胞质着色。结论P物质可能直接作用于破骨细胞,并在正畸牙槽骨改建中发挥作用。     

Effects of 1,25 - （OH） 2 D3 on Stimulation of Osteoclast - like Cells Formationand Expression of ODF mRNA in Murine Marrow Cells in Vitro

目的：观察不同浓度的1，25-(OH)2D3对破骨细胞形成及对骨髓细胞ODFmRNA表达的影响：进一步阐明骨吸收刺激因子在正畸牙周组织改建中的作用。方法：应用不同浓度的1，25-(OH)2D3(0、10^-10、10^-8、10^-6mol／L)诱导大鼠骨髓细胞破骨样细胞的形成，采用体外破骨细胞溶骨模型，观察牙本质片上骨吸收陷窝数目，采用原位杂交技术检测骨髓基质细胞ODF的mRNA表达。结果：随着1，25-(OH)2D3浓度的增加，骨陷窝数明显增多，陷窝面积增大；ODF mRNA表达的阳性信号显著增强。结论：在体外，1，25-(OH)2D3可以调节破骨细胞的骨吸收活性，进而调节局部骨微环境的骨吸收及骨形成平衡的变化。     

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。     

大鼠正畸牙槽骨改建初期转化生长因子β1的表达及意义

目的观察大鼠正畸牙齿移动过程中转化生长因子(TGF-β1)在牙槽骨的表达,探讨其在牙槽骨改建中的调控作用.方法选用25只雄性SD大鼠采用别针簧推上颌切牙向两侧移动的方法,建立牙齿移动及牙槽骨改建模型,并于加力后1d、3d、5d、7d、处死动物取材.用HE染色方法观察牙槽骨的组织学变化,用免疫组织化学方法系列观察牙槽骨中TGF-β1的表达,并检测牙槽骨中增殖细胞核抗原(PCNA)的表达.结果正常牙槽骨组织中,TGF-β1在骨细胞和骨髓组织呈弱阳性表达.牙齿移动3d,压力测牙周膜中前体破骨细胞数量增多,TGF-β1呈阳性表达;张力侧骨髓组织中的TGF-β1免疫反应增强.牙齿移动5d,压力侧的骨吸收陷窝内可见多核破骨细胞,张力侧的牙槽边缘也出现成骨细胞,TGF-β1均呈阳性表达.牙齿移动7d时,张力侧出现少量PCNA呈阳性表达.结论正畸牙齿移动初期,压力侧破骨细胞功能活跃,张力侧面骨细胞的增殖活性随时间逐渐增强.TGF-β1参与了正畸牙槽骨改建初期破骨细胞和成骨细胞的分化形成过程,它是正畸牙槽骨改建中一种重要的局部调控因子.     

白细胞介素1β,肿瘤坏死因子α对牙周组织的作用

目的 :探讨白细胞介素 1β(interleukin - 1β ,IL - 1β)和肿瘤坏死因子α(tumornecrosisfactorα ,TNFα)对牙周组织的作用。方法 :将IL - 1β、TNFα注入到兔的牙周组织 ,以生理盐水为对照 ,不同时期取材做组织学观察和评价。 结果 :实验组 15天后牙龈组织血管扩张充血 ,牙槽骨改建活跃 ,有大量的骨吸收陷窝及破骨细胞 ,并有新骨形成和破骨细胞出现 ;30天后仅表现为大量的骨陷窝及破骨细胞 ,无成骨现象 ,且以联合应用IL - 1β、TNFα组最显著。 结论 :IL - 1β、TNFα均能引起牙龈炎症反应、附着上皮和牙槽骨的破坏 ,提示IL - 1β、TNFα在牙周组织疾病中有作用。     

不同浓度1,25-二羟基维生素D3对体外培养破骨样细胞骨吸收作用的影响

目的:观察不同浓度的1,25-二羟基维生素D3对体外培养破骨样细胞骨吸收作用的影响.进一步阐述骨吸收刺激因子在正畸牙周组织改建中的作用.方法:应用不同浓度的1,25-二羟基维生素D3(0、10-10、10-8、10-6mol/L)诱导大鼠骨髓破骨样细胞的形成,观察细胞在牙本质片上形成骨吸收陷窝的数目以及陷窝面积的大小;并采用原位杂交技术检测大鼠骨髓细胞的ODF mRNA表达.结果:随着1,25-二羟基维生素D3浓度的增加,骨陷窝数明显增多,陷窝面积增大;ODF mRNA表达的阳性信号显著增强.结论:在体外,1,25-二羟基维生素D3可以调节大鼠骨髓破骨样细胞的形成及骨吸收活性.     

重组人血小板衍生生长因子-BB对大鼠正畸牙移动过程中破骨细胞内黏着斑激酶表达的影响

目的观察血小板衍生生长因子-BB（plateletderived growth faetor-BB,PDGF—BB）对大鼠正畸牙移动过程中破骨细胞内黏着斑激酶（focal adhesion kinase,FAK）表达的影响,探讨PDGF-BB对正畸牙移动骨改建中破骨细胞的作用及其下游信号通路。方法80只sn雄性大鼠上颌安装施加50g力的正畸装置,牵引上颌第一磨牙近中移动,隔日于正畸牙局部黏膜注射10ng的重组人血小板衍生生长因子-BB（recombinant human ptatelet derived growth factor-BB,rhPDGF-BB）,对照组注射磷酸盐缓冲液,在加力后1,4、7、10、14d处死动物,制备标本并测量牙齿移动距离,抗酒石酸酸性磷酸酶计数压力侧破骨细胞数量,免疫组织化学方法观察破骨细胞内FAK表达变化。结果PDGF-BB明显促进压力侧破骨细胞增殖,加速正畸牙移动,除第1天实验组与对照组牙移动距离差异无统计学意义以外,其余均有统计学意义（p〈0．05）;各观察点实验组破骨细胞FAK表达均高于对照组,灰度值比较差异均有统计学意义（p〈0.05）。结论外源性的PDGF—BB影响正畸牙移动骨改建过程。在大鼠正畸牙压力侧牙槽骨改建的过程中,FAK参与了破骨细胞信号转导的下游通路,其表达能被外源性的rhPDGF-BB所调节。     

实验性正畸牙齿移动中RANKL在牙周组织中表达的研究

目的 观察实验性正畸牙齿移动过程中，大鼠牙周组织中转录因子NFKB受体活化剂配体（receptor activator of NF-kB ligand／RANKL）的表达及定位，进一步探讨RANKL的来源、作用机制以及与正畸牙齿移动中骨改建的关系。方法 采用SP免疫组织化学方法检测RANKL在正畸大鼠牙周组织中的表达，并采用计算机图像分析的方法对各组RANKL的表达强度进行半定量分析。结果 实验性正畸牙齿移动过程中：①RANKL主要表达于牙槽骨表达的成骨细胞、破骨细胞和牙周膜成纤维细胞胞浆和胞膜中。②RANKL在牙周组织内的表达和分布在时间上是不均衡的，随时间的增加呈先增高后回降的趋势，峰值在第5天。③RANKL在牙周组织内的表达和分布在部位上是不均衡的，压力侧的表达量高于张力侧，牙槽骨表面的表达量高于近牙骨质侧的牙周膜。结论 RANKL的表达与正畸牙槽骨改建过程中破骨细胞的生命过程密切相关，RANKL通过旁分泌、自分泌机制作用于破骨细胞前体、成熟破骨细胞膜表达的受体RANKL，促进破骨细胞前体的分化、成熟，激活成熟破骨细胞的功能，加速骨改建。     

正畸牙齿移动中破骨细胞活动的影响因素

正畸牙齿的移动依赖于牙周组织的改建,其中牙槽骨的改建主要依赖于破骨细胞和成骨细胞的功能活动。破骨细胞来源于造血干细胞,是一种高度分化的多核巨细胞,是正畸牙齿移动过程中压力侧骨吸收的主要功能细胞。破骨细胞的数量及功能是决定正畸牙齿移动效率的主要影响因素之一,其分化受各种全身性因素或局部因素直接或间接的影响,从而影响骨吸收改建,如激素水平,服用药物,局部加载的正畸力量,牙周状态以及各种细胞因子等,研究这些影响因素对于临床加速牙齿移动或增加支抗,减少不必要的牙齿移动均有一定的指导意义。     

大鼠正畸压力侧牙槽骨改建中RANKL和OPG mRNA的表达

目的研究破骨细胞核因子kB受体活化因子配基（receptor activator nuclear factor kappa B ligand,RANKL）及其伪受体骨保护因子（osteoprotegerin,OPG）的mRNA在大鼠正畸牙移动压力侧牙槽骨改建中的表达变化及时问分布特点。方法选用80只6周龄SD雄性大鼠建立正畸牙移动模型,分别在加力后2d、5d、7d、10d和14d各处死16只大鼠。HE染色观察大鼠牙周组织的形态学变化;TRAP染色计数压力侧牙槽骨组织中的破骨细胞数量;实时定量PCR方法检测RANKL和OPGmRNA的表达变化及时问分布特点。结果骨改建的最活跃期为正畸加力后的第7d,压力侧牙槽骨组织中的TRAP染色阳性破骨细胞计数随加力时间的增加而增加,第7d达到高峰,而后逐渐降低。压力侧牙槽骨组织中的RANKL和OPGmRNA表达水平均随加力时间的增加而增加,第7d达到高峰,而后均逐渐降低。结论RANKL和OPG mRNA表达的变化规律不仅与骨改建过程一致,而且也与TRAP染色阳性破骨细胞数量的变化规律一致。RANKL和OPG与正畸牙移动骨改建过程中破骨细胞的分化、形成和功能密切相关。     

Autocrine regulation of osteoclast formation and bone resorption by IL-1 alpha and TNF alpha.

Bone resorption is regulated by the cytokines within marrow cells that mediate osteoclast formation and activation. IL-1 and TNF induce bone resorption by stimulating the production of osteoclast-like multinucleated cells and by increasing the bone-resorbing activity of formed osteoclasts. This study was designed to detect IL-1 and TNF in osteoclasts in vitro and to determine whether these cytokines up-regulate osteoclast differentiation and bone resorption. The production of IL-1 alpha, -beta, and TNF alpha, beta in osteoclasts was examined immunohistochemically and by in situ hybridization. In the co-culture of C57BL/6N mouse bone marrow and MC3T3-G2/PA6 cells, a colony of osteoclasts formed, and IL-1 alpha and TNF alpha were detected. However, IL-1 beta and TNF beta were not detected. To investigate the role of IL-1 alpha and TNF alpha from osteoclasts, we enumerated TRAP-positive cells and measured the resorption pit areas in the presence of antibodies against IL-1 alpha and TNF alpha. The addition of antibodies against IL-1 alpha and TNF alpha to the co-culture system decreased the number of TRAP-positive colonies at seven days after incubation (anti-IL-1 alpha, 25.0 +/- 2.3%; anti-TNF alpha, 41.7 +/- 3.7%; anti-IL-1 alpha + anti-TNF alpha, 40.5 +/- 1.3%; and control, 100%), and the ratio of mononuclear to multinuclear cells had changed (anti-IL-1 alpha, 90:10; anti-TNF alpha, 75:25; anti-IL-1 alpha+ anti-TNF alpha, 88:12; and control, 60:40). The total pit areas per dentin slice also decreased with the addition of antibodies (anti-IL-1 alpha, 28,828; anti-TNF alpha, 49,249; anti-IL-1 alpha + anti-TNF alpha, 30,685; and control, 303,139 mm2). These results suggest that local production of IL-1 alpha and TNF alpha by osteoclasts is an important mechanism for regulating the osteoclast differentiation and bone resorptive process.     

可溶性白细胞介素-1和肿瘤坏死因子受体对大鼠正畸牙移动影响的研究

目的研究局部注射重组人类可溶性受体对大鼠正畸牙移动进程的影响。方法选取64只雄性SD大鼠,从加力第1天起在大鼠牙齿局部分别注射重组人类可溶性白细胞介素- 1受体Ⅱ（sIL- 1- RⅡ）、可溶性肿瘤坏死因子受体Ⅰ（sTNF- RⅠ）、两者混合物,测量磨牙移动距离,用苏木精- 伊红（HE）染色观察被移动磨牙牙周组织形态学变化,并应用抗酒石酸盐酸性磷酸酶（TRAP）组织化学染色对破骨细胞和破牙骨质细胞的数量及分布的时相性改变进行分析。结果各受体组大鼠磨牙牙周组织形态学时相性变化与对照组相似,但各时间点牙槽骨吸收程度均较轻微,多数大鼠牙根表面很少或几乎没有明显牙骨质吸收迹象。与对照组相比,在第14天所有受体注射组牙槽骨及牙根表面TRAP染色阳性细胞降低了约50%（P<0.05）,但受体组组间差异无统计学意义（P>0.05）。结论在大鼠正畸牙局部注射重组人类可溶性白细胞介素- 1和肿瘤坏死因子受体,可以减少牙齿移动距离并减少牙根吸收。     

白细胞介素-6对破骨细胞骨吸收效应及MMP-3表达影响的实验研究

目的：观察不同浓度IL－6对破骨细胞骨吸收的剂量效应及对破骨细胞基质金属蛋白酶－3表达的影响，以期进一步阐明IL－6介导基质金属蛋白酶在破骨细胞性骨吸收中的病理机制。方法：采用体外破骨细胞溶骨模型，通过原子吸收分光光度仪及免疫组化染色技术检测不同浓度IL－6对破骨细胞溶骨活性及其质金属蛋白酶－3表达的影响。结果：当IL－6＞10U／ml时，培养上清中Ca^2 浓度显著增加，牙本质片上骨吸收陷窝数目明显增多；当IL－6浓度为100U／ml、500U／ml时破骨细胞基质金属蛋白酶－3表达的阳性信号显著增强。结论：在IL－6作用下，破骨细胞表达基质金属蛋白酶－3。IL－6对破骨细胞具有激活作用，低浓度主要诱导破骨细胞形成，较高浓度刺激破骨细胞的溶骨活性。     

Diabetes enhances periodontal bone loss through enhanced resorption and diminished bone formation

Using a ligature-induced model in type-2 Zucker diabetic fatty (ZDF) rat and normoglycemic littermates, we investigated whether diabetes primarily affects periodontitis by enhancing bone loss or by limiting osseous repair. Diabetes increased the intensity and duration of the inflammatory infiltrate (P < 0.05). The formation of osteoclasts and percent eroded bone after 7 days of ligature placement was similar, while four days after removal of ligatures, the type 2 diabetic group had significantly higher osteoclast numbers and activity (P < 0.05). The amount of new bone formation following resorption was 2.4- to 2.9-fold higher in normoglycemic vs. diabetic rats (P < 0.05). Diabetes also increased apoptosis and decreased the number of bone-lining cells, osteoblasts, and periodontal ligament fibroblasts (P < 0.05). Thus, diabetes caused a more persistent inflammatory response, greater loss of attachment and more alveolar bone resorption, and impaired new bone formation. The latter may be affected by increased apoptosis of bone-lining and PDL cells.     

Peripheral Blood Mononuclear Phagocytes From Patients With Chronic Periodontitis Are Primed for Osteoclast Formation

Background: During inflammatory periodontal disease, peripheral blood mononuclear cells (PBMCs) are attracted to bone and differentiate into active bone‐resorbing osteoclasts (OCs), thus providing evidence that the impact of chronic periodontitis (CP) on the activity of circulating mononuclear cells is of central importance. The authors test the hypothesis that peripheral blood mononuclear phagocytes (PBMPs) from patients with CP are activated and more susceptible to differentiation into OCs, which in turn would lead to more intense bone resorption. Methods: In vitro cytokine production by both unstimulated and lipopolysaccharide‐stimulated PBMCs from individuals with (n = 10) or without (n = 12) periodontitis was determined by cytokine array. OC differentiation from CD14⁺ PBMCs was induced by receptor activator of nuclear factor‐kappa B ligand (RANKL), either alone or in the presence of macrophage colony‐stimulating factor (M‐CSF). PBMC differentiation to OCs was confirmed by tartrate‐resistant acid phosphatase staining; bone resorbing activity was assessed by using an osteologic plate assay (bone resorption pit formation). Results: PBMCs from patients with CP produced tumor necrosis factor‐α and higher amounts of interferon‐γ, interleukin (IL)‐1α, IL‐1β, IL‐1rα, CXC motif chemokine 10, macrophage migration inhibitory factor, macrophage inflammatory protein (MIP)‐1α, and MIP‐1β than the control cells. OC differentiation was induced by RANKL alone in PBMCs from patients with CP, but not in PBMCs from the healthy controls, which required the addition of M‐CSF. In addition, PBMC‐derived OCs from patients with CP showed significantly higher resorption activity than that observed in the healthy controls. Also, the circulating concentrations of M‐CSF were significantly higher in patients with CP than in the control participants. Conclusions: These data indicate that in patients with CP, circulating PBMCs are primed for increased proinflammatory activity and that M‐CSF plays a central role in this process by increasing OC formation and the consequent bone resorption activity.     

Histomorphometric analysis of osteoclastic resorption in bone directly invaded by gingival squamous cell carcinoma

Whole frontal sections of the mandibular bone from 18 patients with gingival squamous cell carcinoma showing direct bone invasion were prepared for histometrical analysis by a non-decalcified grinding method. The osteoclast cytoplasmic area in tumor sites was larger and its relative frequency distribution was broader than that of osteoclasts in non-tumor sites. The index of bone resorption was significantly increased, while that of bone formation was decreased in the tumor sites compared to the non-tumor sites ( P <0.01). In the tumor sites, the indices of bone resorption and formation in the group showing greatest chemotherapy and radiation therapy effects (CRE) were significantly different from those in the group with low CRE ( P <0.01). A history of irradiation therapy was associated with significantly decreased bone resorption ( P <0.05).     

淫羊藿苷、黄芩苷和大黄素联合作用对体外骨吸收的影响

目的研究淫羊藿苷、黄芩苷和大黄素单独及联合作用对体外骨吸收的影响。方法将从新生兔长骨分离的细胞与牙本质磨片共培养,建立体外骨吸收模型,并加入白细胞介素(IL)-1β及淫羊藿苷0.01μg/ml、黄芩苷0.01μg/ml、大黄素10μg/ml或其等比组合进行干预,动态观察并测量吸收陷窝的数量和面积。结果分离的细胞与牙本质片共同培养24 h后,牙本质片上出现少量吸收陷窝。体外共培养10 d,加入IL-1β的阴性对照组的陷窝数量和面积大于空白对照组(P<0.05)。同时加入提取物的各组陷窝数量和面积均小于阴性对照组(P<0.05)。其中,淫羊藿苷组、黄芩苷组和大黄素组3组之间无显著差异(P>0.05),而组合组陷窝数量和面积均小于上述3组(P<0.01)。结论淫羊藿苷0.01μg/ml、黄芩苷0.01μg/ml、大黄素10μg/ml等比组合可抑制IL-1β诱导的骨吸收。     

The role of RANKL and MMP‐9 in the bone resorption caused by ameloblastoma

J Oral Pathol Med (2010) 39 : 592–598 Background: Ameloblastoma, a common odontogenic tumor located in jaws, generally leads to severe damage to patient’s complexion and masticatory function. To expand in jaws, ameloblastoma must have a mechanism of resorbing the surrounding bone. Our objective was to explore the bone‐resorption mechanism of ameloblastoma by observing the role of Receptor activator of nuclear factor kappa B ligand (RANKL) and matrix metalloproteinase‐9 (MMP‐9) in the bone‐resorption process. Methods: In the study, the expression of RANKL and MMP‐9 in ameloblastoma was detected using immunohistochemistry (IHC) and RT‐PCR. Then, co‐culture system of ameloblastoma cells and bone marrow cells from neonatal rabbit was erected to observe the potential of ameloblastoma cells to induce osteoclastogenesis. Finally, the induced osteoclasts were used for in vitro bone‐resorption assay. In the co‐culture system and the bone‐resorption assay, the selective inhibitor of RANKL and MMP‐9, osteoprotegerin (OPG) and tissue inhibitor of metalloproteinase‐1 (TIMP‐1) were, respectively, used for observing the role of RANKL and MMP‐9. Results: The expression of RANKL and MMP‐9 in ameloblastoma was confirmed. Ameloblastoma cells were found to induce bone marrow cells from neonatal rabbit differentiate into osteoclasts with bone‐resorption activity. In addition, OPG was found to, respectively, have markedly inhibitory effect on osteoclastogenesis (P < 0.01), and slightly inhibitory action on bone resorption (P < 0.05). Conclusions: Ameloblastoma cells had the potential to induce osteoclastogenesis. Moreover, RANKL played an essential role in the in vitro osteoclast formation and bone resorption induced by ameloblastoma cells.     

髓样细胞触发受体家族与牙周炎病因机制的研究进展

牙周炎是由多种微生物与宿主免疫反应引起的慢性炎症性疾病,主要累及牙龈、牙周膜、牙槽骨等牙周支持组织。髓样细胞触发受体(triggering receptors expressed on myeloid cells, TREMs)是近年发现的一类细胞膜受体家族,其中髓样细胞触发受体-1(triggering receptors expressed on myeloid cells-1,TREM-1)主要表达于单核/巨噬细胞、中性粒细胞和树突细胞等骨髓分化细胞表面,也在血管平滑肌、上皮细胞中有表达。TREM-1、可溶性髓系细胞触发受体-1(soluble triggering receptors expressed on myeloid cells-1,sTREM-1)表达可被牙龈卟啉单胞菌等牙周病致病菌正向调控。髓样细胞触发受体-2(triggering receptors expressed on myeloid cells-2,TREM-2)在破骨细胞形成过程中起重要作用,可能与牙周炎导致的牙槽骨吸收有关。本文讨论了牙周炎病因机制的研究进展,以及TREMs家族在其中发挥的作用。