【作者】 白玉娣；

【导师】 杨富生；

【作者基本信息】 中国人民解放军第四军医大学 ， 口腔临床医学， 2003， 硕士

【摘要】 肿瘤坏死因子家族成员骨保护素配体(osteoprotegerin ligand，OPGL；又称TRANCE，RANKL，ODF)是近年发现的在动物的骨、血液及免疫系统等处分布的，促进破骨细胞分化的蛋白分子。在骨改建中，诱导破骨细胞融合、分化、成熟等一系列功能活动。核因子κB受体活化剂(receptor activator of nuclear factor-κB，RANK)是肿瘤坏死因子受体超家族成员，是OPGL在破骨细胞表面的受体，OPGL通过与其结合，将细胞分化信号传入破骨细胞前体细胞内，促进后者增殖、分化、融合和成熟破骨细胞的激活。骨保护素(osteoprotegerin，OPG；又称为OCIF)是肿瘤坏死因子家族成员，对破骨细胞生理性骨吸收起着关键的调控作用。OPG由成骨细胞和骨髓基质细胞产生，与其自然配体OPGL结合阻断了OPGL激活其同族受体RANK所产生的促破骨细胞分化、活化和存活作用，转基因小鼠过度分泌OPG可导致因破骨细胞几乎完全丧失引起的骨质硬化症。OPGL、RANK、与OPG共同构成一种细胞因子系统，调节破骨细胞的增殖、分化、激活和骨吸收功能等生物学活性，对有机体的成骨和破骨作用进行调节。目前对该三类分子的研究主要集中于骨的改建方面，而其在乳恒牙替换过程中所起作用的研究还未见报道。乳恒牙替换是动物牙齿及颌面部生长发育的重要阶段，包含有乳牙根、牙槽骨的吸收、恒牙胚的发育和恒牙萌出等一系列复杂的生理过程。在此过程中，乳牙、牙槽骨、牙周膜、恒牙胚等组织都发生了一系列复杂的变化。本研究采用免疫组织化学染色、原位杂交、细胞培养等方法，首次观察了犬乳恒牙替换过程中，乳牙牙根稳定期、乳牙根吸收期、恒牙列期三个阶段，乳牙牙根、牙周膜、牙槽骨和恒牙胚中OPGL及其受体RANK的表达染色情况，以及破骨细胞培养分化过程中OPGL和RANK的表达情况，试图初步探讨在乳恒牙替换期和破骨细胞体外培养中这两类因子起到怎样的作用。 第四军医大学硕士学位论文主要研究内容及结果如下： 第一部分 犬乳恒牙替换阶段OPGL表达的研究 分别采用免疫组化和原位杂交方法，观察犬乳牙根稳定期、乳牙根生理性吸收期和恒牙列期中OPGL的表达染色情况。 1．犬乳牙牙根稳定期：犬乳牙牙根尚未发生生理性根吸收，恒牙胚处于发育阶段，恒牙胚牙本质、牙釉质基质开始形成，牙槽骨处于发育塑形期，此时牙槽骨成骨细胞、骨陷窝内破骨细胞、恒牙胚的成釉细胞、成牙本质细胞免疫组化染色阳性，同时发现恒牙胚釉基质也为阳性着色；成牙本质细胞、牙槽骨成骨细胞原位杂交结果阳性。提示成骨细胞表达OPGL，作用于破骨细胞，在根稳定期参与牙槽骨的发育塑形，成牙本质细胞表达OPGL，可能参与恒牙胚的发育过程。 2．犬乳牙牙根吸收期：犬乳牙牙根发生生理性吸收，乳牙根面蚕食状，可见多数吸收陷窝，陷窝中可见多核破牙细胞。牙囊周围及接近牙胚的牙槽骨陷窝内可见多核破骨细胞。恒牙胚冠部牙本质、牙釉质进一步形成和矿化，其成釉细胞、成牙本质细胞高柱状，胞核远离基底膜，极性倒置明显。此时可见牙槽骨成骨细胞、破骨细胞、恒牙胚的成釉细胞、成牙本质细胞和釉基质免疫组化染色阳性，并且发现多核破牙细胞也为阳性着色。其中破骨细胞、成骨细胞染色灰度值较稳定期显著降低0O刀5人提示此期 OPGL染色强度明显升高。成牙本质细胞、牙槽骨成骨细胞原位杂交结果阳性，且成骨细胞灰度值较根稳定期显著降低O川刀5L提示此期成骨细胞 OPGL分泌功能增强，参与破骨细胞和破牙细胞的分化成熟，导致牙槽骨和乳牙根的吸收。成牙本质细胞分泌OPGL，可能参与恒牙胚的发育。 3犬恒牙列期：乳牙脱落，恒牙萌出。牙槽骨破骨细胞免疫组化染色弱阳性，成骨细胞原位杂交结果阳性。两类细胞染色灰度值均显著增加0 （0刀 1），提示成骨细胞 OPGL分泌量明显减少，牙槽骨的塑形可能成骨作 ·6－ 第四军医大学硕士学位论文用占优势。 第二部分 犬乳恒牙替换阶段oWK表达的研究 本实验采用免疫组化方法，观察RAN’K在犬乳牙根稳定期、生理性吸收期和恒牙列期中的表达染色情况。 1．犬乳牙牙根稳定期：可见牙槽骨陷窝内破骨细胞免疫组化阳性，并发现恒牙胚成牙本质细胞阳性着色。提示破骨细胞表达RAN’K，参与牙槽骨的改形重建，成牙本质细胞表达RANK，可能参与恒牙胚的发育。 2犬乳牙牙根吸收期：乳牙根发生生理性吸收，牙槽骨陷窝内破骨细胞、恒牙胚成牙本质细胞染色阳性，并发现根面吸收陷窝中的破牙细胞亦为阳性着色。其中破骨／牙细胞为强阳性着色，破骨细胞灰度值较其他两组显著降低0N刀5人 表明RAN’K染色强度明显升高，提示破骨／牙细胞RAN’K分泌量显著增加，参与牙槽骨和乳牙根的吸收功能增强，恒牙胚成牙本质细胞分泌RANK，参与恒牙胚发育。 3．犬恒牙列期：乳牙脱落，恒牙萌出。牙槽骨内破骨细胞染色弱阳性，灰度值较前两期显著升高（P（0．of），提示破骨细胞RAN’K?更多还原

【Abstract】 The rumor-necrosis-factor-family molecule osteoprotegerin ligand(OPGL; also known as TRANCE,RANKL and ODF)has been lately identified as a potential osteoclast differentiation factor that distributes in bone,blood and immune system.During bone remodeling,it can induce osteoclasts fusion, differentiation,maturation and so on.OPGL plays diverse roles in different tissues. The receptor activator of nuclear factor K B(RANK)was identified as a TNF super family member.It is the receptor of OPGL on osteoclast precursor cells.OPGL binds specifically to RANK and transfers the potential osteoclastogenic signals such as cell differentiation into osteoclast precursor cells to stimulate the latter fusing and activating.Osteoprotegerin(OPG) is also a new member of TNF receptor family which plays a key role in the physiological regulation of osteoclastic bone resorption.The protein,which is produced by osteoblasts and marrow stromal cells,acts by binding to its natural ligand OPGL and this binding prevents OPGL from activating its cognate receptor RANK,which is the osteoclast receptor vital for osteoclast differentiation,activation and survival. Overexpression of OPG in transgenic mice leads to profound osteopetrosis secondary to a near total lack of osteoclasts.OPGL-RANK-OPG comprises a keycytokone system that governs osteoclast develoment and regulates bone morphogenesis and resorption.At present,the study of the three cytokines focuses on bone remodeling.The replacement of primary and permanent teeth is an important part that belongs to the growth and development of animals’ teeth and maxillofacial region.A lot of changes have taken place in the correlated tissues including the primary teeth,alveolar bone,periodontal ligament and the successional permanent teeth germs during this procedure.Until now,there is not any report concerning about the roles of the three cytokines in the replacement of the primary and permanent teeth.So in our study,we observed OPGL and its’ receptor RANK expressions in deciduous teeth roots,periodontal ligament, alveolar bone and permanent teeth germs under three states of the dogs’ teeth (primary dentition roots stability,primary dentition roots resorption,permanent dentition)by immunohistochemical technique,in situ hybridization method and cell culture.The main content and results of our researches are below:Parti OPGL expression during the replacement of dogs’ primary and permanent teethThere were two experiments in part 1.Immunohistochemical technique and in situ hybridization method were used in this study to observe the OPGL expression under three states of the dogs’ teeth: primary dentition roots stability,primary dentition roots resorption, permanent dentition.1.1 dogs’ primary dentition roots stability stage:The deciduous teeth roots were not absorbed biologically.The permanent teeth germs were developing and dentin and enamel matrix started to form.In the immunohistochemical experiment, OPGL positive signals were seen in the multinucleated osteoclasts localized in the absorbed lacunae of the alveolar bone near the germs,as well as ameloblasts, odontoblasts of the permanent teeth germs. We also detected that enamal matrix of the permanent teeth germs dyed brown in the experimentln the in situ hybridization experiment, we saw that OPGL-positive cells were identified as osteoblasts in the alveolar bone and odontoblasts of the germs.These resultsindicated that osteoblasts and odontoblasts expressed OPGL and OPGL involved in the development of dogs’ permanent teeth germs during the stages of the primary dentition roots stability.1.2 dogs’ primary dentition roots resorption stage :Dogs’ deciduous teeth roots were undergoing absorbed biologically and there were a lot of absorbed lacunae on the roots.Dentin and enamel of the permanent teeth germs went on forming and calcifying.Ameloblasts appeared to tall posts and the nuclears were away from the membranes.In the immunohistochemical experiment,the OPGL-positive signals were observed on osteoclasts in更多还原