犬乳恒牙替换期骨保护素配体和核因子κB受体活化剂表达的研究
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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?
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 key
cytokone 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 teeth
There 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 results
indicated 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
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?
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 key
cytokone 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 teeth
There 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 results
indicated 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
引文
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29. Darnay BG,Ni J,Moore PA.et al.Activation of NF-κB by RANK requires tumor necrosis factor receptor-associated factor(TRAF)6 and NF-κ B-inducing kinase. Identification of a TRAF6 interaction motiff. J Bio Chem 1999;274:7724～7731.
30. Hofbauer LC,Khosla S,Lacey DL.et al.The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res.2000; 15:2～12.
31. Nakashima T, Kobayashi Y, Yamasaki S.et al.Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappa B ligand:modulation of the expression by osteotropic factors and cytokines. Biochem Biophys Res Commun. 2000;275(3): 768～775.
32. Aubin JE,Bonnelye E.Osteoprotegerin and its ligand: a new paradigm for regulation of osteoclastogenesis and bone resorption. Medscape Womens Health. 2000;5(2):5.
33. Shevde NK,Bendixen AC,Dienger KM.et al. Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression. Proc Natl Acad Sci.USA.2000; 97(14):7829～7834.
34. Mukohyama H,Ransjo M,Taniguchi H.et al.The inhibitory effects of vasoactive intestinal peptide and pituitary adenylate cyclase-activafing polypeptide on osteoclast formation are associated with upregulation of osteoprotegerin and downregulation of RANKL and RANK.Biochm Biophys Res Commun. 2000;271:158～163.
35. Oyajobi BO,Anderson DM,Traianedes K.et al.Therapeutic efficacy of a soluble receptor activator of nuclear factor kappaB-IgG Fc fusion protein in suppressing bone resorption and hypercalcemia in a model of humoral hypercalcemia of malignancy. Cancer Res.2001; 61(6): 2572～2578.
36. Yasuda H,Shima N,Nakagawa N.et al. Identify of osteoclastogenesis inhibitory factor(OCIF)and osteoprotegerin(OPG):a mechenism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology. 1998; 139:1329～1337.
37. Hofbauer LC,Heufelder AE.The role of osteoprotegerin and osteoprotegerin ligand in the pathogenesis and treatment of metabolic bone diseases.J Clin Endocrinol Metab.2000;85:2355～2363.
38. Burgess TL,Qian Y, Kaufman S,et al.The ligand for osteoprotegerin(OPGL) directly activates mature osteoclasts.J Cell Biol. 1999; 145:527～538.
39. Jimi E,Akiyama S,Tsumkai T, et al.Osteoclastdifferentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function.J Immunol. 1999;163:434～442.
40. Josien R, Wong BR, Li H-L,et al.TRANCE,a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendric cells. J Immunol. 1999; 162:2562～2568.
41. Wong BR,Josien R, Choi Y. TRANCE is a TNF family member that regulates dedritic cell and osteoclast function. J Leukoc Biol. 1999;65:715～724.
42. Faucheux C, Horton MA, Price JS. Nuclear localization of type Ⅰ parathyroid hormone/parathyroid hormone-ralated protein receptors in deer antler osteoclasts: evidence for parathyroid hormone-ralated protein and receptor activator of NF-kappaB-dependent effects on osteoclast formation in regenerating mammalian bone.J Bone Miner Res.2002;17(3):455～464.
43. Min H,Morony S,Sarosi I,et al. Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a
process resembling osteoclastogenesis.J Exp Med.2000;192(4):463～474.
44. Mukohyama H,Ransjo M,Taniguchi H.et al.The inhibitory effects of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide on osteoclast formation are associated with upregulation of osteoprotegerin and downregulation of RANKL and RANK.Biochemical and Biophysical Research Communications. 2000;271:158～163.
45. Ueland T, Bollerslev J,Mosekilde L.Osteoclast function is regulated by neighbouring osteoblasts. Osteoprotegerin, RAND and RANK ligand constitute a unique regulatory for bone resorption with important pathophysiological and therapeutic aspects.Ugeskr Laeger. 2002;164(27):3526～3530v.
46. Khosla S. Minireview: the OPG/RANKL/RANK system.Endocrinology.2001; 142(12):5050～5055.
47. Hofbauer LC,Neubauer A,Heufelder AE.Receptor activator of nuclear factor-kappa B ligand and osteoprotegerin: potential implications for the pathogenesis and treatment of malignant bone diseases. Cancer.2001 ;92(3):460～470.
48．吴奇光口腔组织病理学 人民卫生出版社 1994
49. Oral Histology and embryology. London:The C.V. Mosby Company 1980;371～403
50. Domon T. Mononuclear odontoclast participation in tooth resorption:the distribution of nuclei in human odontoclasts.Anat Res, 1997 Dec;249(4):449～57
51. Matsuda E.Ultrastructural and cytochemical study of the odontoclasts in physiologic root resorption of human deciduous teeth. J Electron Microsc. 1992,41:131～140.
52. Sasaki T, Shimizu T, Suzuki H,et al. Cytodifferentiation and degeneration of odontoclasts in physiologic root resorption of kitten deciduous teeth. Acta Anat. 1989,135: 330～340
53. Sasaki T, Shimizu T, Watanabe C,et al. Cellular roles in physiological root resorption of deciduous teeth in the cat.J Dent Res. 1990,69:67～74.
54. Sahara N,Ashizawa Y, Nakamura K,et al. Ultrastructural features of odontoclasts that resorb enamel in human deciduous teeth prior to shedding.Anat Rec. 1998,252:215～228.
55. Sahara N,Okafuji N,Toyoki A,et al.Odontoclastic resorption of the superficial nonmineralized layer of predentine in the shedding of human deciduous teeth. Cell Tissue Res. 1994,277:19～26.
56．宇田川信之，高桥直之，须田立雄．破骨细胞形成仕组分子解明．齿界展望，1998；92(1)：5～10
57. Zhou D.Histomorphometric and biochemical study of osteoclast at orthodontic compression sites in the rat during indometracin inhibition. Arch Oral Biol,1997;42(10～11): 717～26
58. Sasaki T, Motegi N,Suzuki H,et al. Dentin resorption mediated by oeontoclasts in physiological root resorption of human deciduous teeth. Am J Anat. 1988, 183:303～315.
59. Matsuda E.Ultrastractural and cytochomical study of the odontoclast in physiologic root resorption of human deciduous teeth. J Electron Microsc Tokyo, 1992;41 (3): 131～40
60. Sasake T. Cytodifferentiation and degeration of odontoclasts in physiologic root resorption of kitten deciduous teeth. Acta Anat Basel. 1989; 135(4):330～40
61．王洪复 骨细胞图谱与骨细胞体外培养技术．2001．
62. Wise GE, Lumokin S J, Huang H, et al. Osteoprotegerin and osteoclast differentiation factor in tooth eruption.J Dent Res.2000;79(12): 1937～1942.
63. Wise GE, Frazier-Bowers S, D'Souza RN. Cellular, molecular, and genetic determinents of tooth eruption. Crit Rev Oral Biol Med.2002; 13 (4): 323～335.
64. Wise GE,Yao S,Zhang Q.et al. Inhibition of osteoclastogenesis by the
secretion of osteoprotegerin in vitro by rat dental follicle cells and its implications for tooth eruption. Arch Oral Biol.2002;47(3):247～254.
65. Wise GE,Grier RL,Lumpkin SJ,et al.Effects of dexamethasone on tooth eruption in rats: differences in incisor and molar eruption. Clin Anat.2001; 14(3):204～209.
66. Shiotani A, Shibasaki Y, Sasaki T. Localization of receptor activator of NfkappaB ligand,RANKL,in periodontal tissues durong experimental movement of rat molars. J Electron Microsc.2001;50(4): 365～369.
67. Oshiro T, Shibasaki Y, Martin TJ,et al. Immunolocalization of vacuolar-type H~+-ATPase,cathepsin K,matrix metalloproteinase-9,and receptor activator of NfkappaB ligand in odontoclasts during physiological root resorption of human deciduous teeth. Anat Rec.2001 ;264(3):305～311.
68. Rani CS, MacDougall M. Dental cells express factors that regulate bone resorption. Mol Cell Biol Res Commun.2000;3(3): 145～152.
69. Jalevik B, Fasth A, Dahllof G. Dental development after successful treatment of infantile osteopetrosis with bone marrow transplantation. Bone Marrow Transplant.2002 ;29(6):537～540.
70. Min H, Morony S, Sarosi I. Osteoprotegerin reverses osteoporosis by inhibiting endoosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis.J Exp Med.2000; 192(4):463～474.
71. Kim N,Odgren PR, Kim DK,et.al.Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene. Proc Natl Acad Sci USA.2000;97(20): 10905～10910.
72. Kostenuik PJ, Shalhoub V. Osteoprotegerin: a physiological and pharmacological inhibitor of bone resorption. Curr Pharm Des 2001;7(8):613～635.
73. Kanzaki H, Chiba M, Shimizu Y, et al. Dual regulation of osteoclast differentiation by periodontal ligament cells through RANKL stimulation and OPG inhibition.J Dent Res.2001; 80(3): 887～891.
74. Kanzaki H, Chiba M, Shimizu Y, et al. Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB up-regulation via prostaglandin E2 synthesis. J Bone Miner Res. 2002;17(2):210～220.
75. Kawamoto S, Ejiri S, Hoshi K, et al. Immunolocalization of osteoclast differentiation factor in rat periodontium. Arch Oral Biol.2002; 47(1):55～58.
76. Sasaki T, Shimizu T, Suzuki H, et al. Cytodifferentiation and degeneration of odontoclasts in physiologic root resorption of kitten deciduous teeth. Acta Anat. 1989;135:330～340.
77. Rodan GA,Martin TJ.Role of osteoblasts in hormonal control of bone resorption-a hypothesis. Calcif Tissue Int. 1981 ;33:349.
78. Mundy GR,Roodman GD.Osteoclast ontogeny and function. In: Peck WA Mineral Research. 1987;5:209.
79. Vaes G. Cellular biology and biochemical mechanism of bone resorption. Clin Orthop. 1988;231:239. 4.
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85. White E.Life,death,and the pursuit of apoptosis. Genes Dev. 1996; 10:1～15.
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90．王兴强体外培养大鼠破骨细胞的生物学特性研究第四军医大学学位论文
91．于明香，金慰芳，王洪复等．破骨细胞体外培养与形态观察．上海医科大学学报．1996；23(1)：52～54．
2. Yasuda H,Shima N,Nakagawa N.et al.Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.Proc.Natl.Acad. Sci.USA. 1998;95:3597～3602.
3. Lacey DL,Timms E,Tan HL.et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998;93:165～176.
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13. Liu BY, Guo J,Lanske B.et al. Conditionally immortalized murine bone marrow stromal cells mediate parathyroid hormone-dependent osteoclastogenesis in vitro. Endocrinology. 1998; 139:1952～1964.
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15. Tsurukai T, Takahashi N,Jimi E.et al.Isolation and characterization of osteoclast precursors that differentiates into osteoclasts on calvarial cells within a short period of time.J.Cell.Physiol. 1998; 177:26～35.
16. Fuller K,Wong B,Fox S.et al. TRANCE is necessary sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J.Exp.Med. 1998;188:997～1001.
17. Tsukii K,Shima N,Mochizuki S.et al. Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1,25-dihydroxyvitamin D3,prostaglandin E2,or parathyroid hormone in the microenvironment of bone. Biochem.Biophys.Res. Commun. 1998;246:337～341.
18. Burgess TL,Qian Y, Kaufman S.et al.The ligand for osteoprotegerin(OPGL) directly activates mature osteoclasts. J.Cell.Biol. 1999; 145(3):527～538.
19. Corisdeo S,Gyda M,Zaidi M.et al.New insights into the regulation of cathepsin K gene expression by osteoprotegerin lingand. Biochem. Biophys. Res. Commun.2001 ;285:335～339.
20. Lacey DL, Hong Lin Tan, John Lu, et al. Osteoprotegerin lingand modulates murine osteoclast survival in vitro and in vivo. Am.J.Pathol.2000;157(2):435～448.
21. Nakagawa N,Kinosaki M,Yamaguchi K,et al. RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis.Biochem. Biophys. Res. Commun. 1998;253:395～400.
22. Simonet WS, Lacey DL, Dunstan CR. et al. Osteoprotegerin:a novel secreted protein involved in the regulation of bone density. Cell. 1997;89: 309～319.
23. Greenfield EM,Bi Y, and Miyauchi A.Regulation of osteoclast activity. Life Science. 1999;65:1087～1102.
24. Hsu H,Lacey DL,Dunstan CR.et al.Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc.Natl. Acad. Sci. USA. 1999;96:3540～3545.
25. Arai F, Miyamoto T, Ohneda O. et al. Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-fms and receptor activator of nuclear factor κ B(RANK)receptors.J Exp Med. 1999;190:1741～1754.
26. Yamane T, Kunisada T, Yamazaki H. et al. Sequential requirements for SCL/tal-1,GATA-2,macrophage colony-stimulating factor, and osteoclast differentiation factor/osteoprotegerin ligand in osteoclast development. Exp Hematol. 2000;28:833～840.
27. Smith CA,Farrah T, Goodwin RG. The TNT receptor superfamily of cellular
and viral proteins: activation,costimulation,and death. Cell. 1994;76:959～962.
28. Li J,Sarosi I,Yan XQ.et al.RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc.Natl.Acad. Sci.USA. 2000;97(4):1566～1571.
29. Darnay BG,Ni J,Moore PA.et al.Activation of NF-κB by RANK requires tumor necrosis factor receptor-associated factor(TRAF)6 and NF-κ B-inducing kinase. Identification of a TRAF6 interaction motiff. J Bio Chem 1999;274:7724～7731.
30. Hofbauer LC,Khosla S,Lacey DL.et al.The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res.2000; 15:2～12.
31. Nakashima T, Kobayashi Y, Yamasaki S.et al.Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappa B ligand:modulation of the expression by osteotropic factors and cytokines. Biochem Biophys Res Commun. 2000;275(3): 768～775.
32. Aubin JE,Bonnelye E.Osteoprotegerin and its ligand: a new paradigm for regulation of osteoclastogenesis and bone resorption. Medscape Womens Health. 2000;5(2):5.
33. Shevde NK,Bendixen AC,Dienger KM.et al. Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression. Proc Natl Acad Sci.USA.2000; 97(14):7829～7834.
34. Mukohyama H,Ransjo M,Taniguchi H.et al.The inhibitory effects of vasoactive intestinal peptide and pituitary adenylate cyclase-activafing polypeptide on osteoclast formation are associated with upregulation of osteoprotegerin and downregulation of RANKL and RANK.Biochm Biophys Res Commun. 2000;271:158～163.
35. Oyajobi BO,Anderson DM,Traianedes K.et al.Therapeutic efficacy of a soluble receptor activator of nuclear factor kappaB-IgG Fc fusion protein in suppressing bone resorption and hypercalcemia in a model of humoral hypercalcemia of malignancy. Cancer Res.2001; 61(6): 2572～2578.
36. Yasuda H,Shima N,Nakagawa N.et al. Identify of osteoclastogenesis inhibitory factor(OCIF)and osteoprotegerin(OPG):a mechenism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology. 1998; 139:1329～1337.
37. Hofbauer LC,Heufelder AE.The role of osteoprotegerin and osteoprotegerin ligand in the pathogenesis and treatment of metabolic bone diseases.J Clin Endocrinol Metab.2000;85:2355～2363.
38. Burgess TL,Qian Y, Kaufman S,et al.The ligand for osteoprotegerin(OPGL) directly activates mature osteoclasts.J Cell Biol. 1999; 145:527～538.
39. Jimi E,Akiyama S,Tsumkai T, et al.Osteoclastdifferentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function.J Immunol. 1999;163:434～442.
40. Josien R, Wong BR, Li H-L,et al.TRANCE,a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendric cells. J Immunol. 1999; 162:2562～2568.
41. Wong BR,Josien R, Choi Y. TRANCE is a TNF family member that regulates dedritic cell and osteoclast function. J Leukoc Biol. 1999;65:715～724.
42. Faucheux C, Horton MA, Price JS. Nuclear localization of type Ⅰ parathyroid hormone/parathyroid hormone-ralated protein receptors in deer antler osteoclasts: evidence for parathyroid hormone-ralated protein and receptor activator of NF-kappaB-dependent effects on osteoclast formation in regenerating mammalian bone.J Bone Miner Res.2002;17(3):455～464.
43. Min H,Morony S,Sarosi I,et al. Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a
process resembling osteoclastogenesis.J Exp Med.2000;192(4):463～474.
44. Mukohyama H,Ransjo M,Taniguchi H.et al.The inhibitory effects of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide on osteoclast formation are associated with upregulation of osteoprotegerin and downregulation of RANKL and RANK.Biochemical and Biophysical Research Communications. 2000;271:158～163.
45. Ueland T, Bollerslev J,Mosekilde L.Osteoclast function is regulated by neighbouring osteoblasts. Osteoprotegerin, RAND and RANK ligand constitute a unique regulatory for bone resorption with important pathophysiological and therapeutic aspects.Ugeskr Laeger. 2002;164(27):3526～3530v.
46. Khosla S. Minireview: the OPG/RANKL/RANK system.Endocrinology.2001; 142(12):5050～5055.
47. Hofbauer LC,Neubauer A,Heufelder AE.Receptor activator of nuclear factor-kappa B ligand and osteoprotegerin: potential implications for the pathogenesis and treatment of malignant bone diseases. Cancer.2001 ;92(3):460～470.
48．吴奇光口腔组织病理学 人民卫生出版社 1994
49. Oral Histology and embryology. London:The C.V. Mosby Company 1980;371～403
50. Domon T. Mononuclear odontoclast participation in tooth resorption:the distribution of nuclei in human odontoclasts.Anat Res, 1997 Dec;249(4):449～57
51. Matsuda E.Ultrastructural and cytochemical study of the odontoclasts in physiologic root resorption of human deciduous teeth. J Electron Microsc. 1992,41:131～140.
52. Sasaki T, Shimizu T, Suzuki H,et al. Cytodifferentiation and degeneration of odontoclasts in physiologic root resorption of kitten deciduous teeth. Acta Anat. 1989,135: 330～340
53. Sasaki T, Shimizu T, Watanabe C,et al. Cellular roles in physiological root resorption of deciduous teeth in the cat.J Dent Res. 1990,69:67～74.
54. Sahara N,Ashizawa Y, Nakamura K,et al. Ultrastructural features of odontoclasts that resorb enamel in human deciduous teeth prior to shedding.Anat Rec. 1998,252:215～228.
55. Sahara N,Okafuji N,Toyoki A,et al.Odontoclastic resorption of the superficial nonmineralized layer of predentine in the shedding of human deciduous teeth. Cell Tissue Res. 1994,277:19～26.
56．宇田川信之，高桥直之，须田立雄．破骨细胞形成仕组分子解明．齿界展望，1998；92(1)：5～10
57. Zhou D.Histomorphometric and biochemical study of osteoclast at orthodontic compression sites in the rat during indometracin inhibition. Arch Oral Biol,1997;42(10～11): 717～26
58. Sasaki T, Motegi N,Suzuki H,et al. Dentin resorption mediated by oeontoclasts in physiological root resorption of human deciduous teeth. Am J Anat. 1988, 183:303～315.
59. Matsuda E.Ultrastractural and cytochomical study of the odontoclast in physiologic root resorption of human deciduous teeth. J Electron Microsc Tokyo, 1992;41 (3): 131～40
60. Sasake T. Cytodifferentiation and degeration of odontoclasts in physiologic root resorption of kitten deciduous teeth. Acta Anat Basel. 1989; 135(4):330～40
61．王洪复 骨细胞图谱与骨细胞体外培养技术．2001．
62. Wise GE, Lumokin S J, Huang H, et al. Osteoprotegerin and osteoclast differentiation factor in tooth eruption.J Dent Res.2000;79(12): 1937～1942.
63. Wise GE, Frazier-Bowers S, D'Souza RN. Cellular, molecular, and genetic determinents of tooth eruption. Crit Rev Oral Biol Med.2002; 13 (4): 323～335.
64. Wise GE,Yao S,Zhang Q.et al. Inhibition of osteoclastogenesis by the
secretion of osteoprotegerin in vitro by rat dental follicle cells and its implications for tooth eruption. Arch Oral Biol.2002;47(3):247～254.
65. Wise GE,Grier RL,Lumpkin SJ,et al.Effects of dexamethasone on tooth eruption in rats: differences in incisor and molar eruption. Clin Anat.2001; 14(3):204～209.
66. Shiotani A, Shibasaki Y, Sasaki T. Localization of receptor activator of NfkappaB ligand,RANKL,in periodontal tissues durong experimental movement of rat molars. J Electron Microsc.2001;50(4): 365～369.
67. Oshiro T, Shibasaki Y, Martin TJ,et al. Immunolocalization of vacuolar-type H~+-ATPase,cathepsin K,matrix metalloproteinase-9,and receptor activator of NfkappaB ligand in odontoclasts during physiological root resorption of human deciduous teeth. Anat Rec.2001 ;264(3):305～311.
68. Rani CS, MacDougall M. Dental cells express factors that regulate bone resorption. Mol Cell Biol Res Commun.2000;3(3): 145～152.
69. Jalevik B, Fasth A, Dahllof G. Dental development after successful treatment of infantile osteopetrosis with bone marrow transplantation. Bone Marrow Transplant.2002 ;29(6):537～540.
70. Min H, Morony S, Sarosi I. Osteoprotegerin reverses osteoporosis by inhibiting endoosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis.J Exp Med.2000; 192(4):463～474.
71. Kim N,Odgren PR, Kim DK,et.al.Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene. Proc Natl Acad Sci USA.2000;97(20): 10905～10910.
72. Kostenuik PJ, Shalhoub V. Osteoprotegerin: a physiological and pharmacological inhibitor of bone resorption. Curr Pharm Des 2001;7(8):613～635.
73. Kanzaki H, Chiba M, Shimizu Y, et al. Dual regulation of osteoclast differentiation by periodontal ligament cells through RANKL stimulation and OPG inhibition.J Dent Res.2001; 80(3): 887～891.
74. Kanzaki H, Chiba M, Shimizu Y, et al. Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB up-regulation via prostaglandin E2 synthesis. J Bone Miner Res. 2002;17(2):210～220.
75. Kawamoto S, Ejiri S, Hoshi K, et al. Immunolocalization of osteoclast differentiation factor in rat periodontium. Arch Oral Biol.2002; 47(1):55～58.
76. Sasaki T, Shimizu T, Suzuki H, et al. Cytodifferentiation and degeneration of odontoclasts in physiologic root resorption of kitten deciduous teeth. Acta Anat. 1989;135:330～340.
77. Rodan GA,Martin TJ.Role of osteoblasts in hormonal control of bone resorption-a hypothesis. Calcif Tissue Int. 1981 ;33:349.
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