【作者】 陈旭；

【导师】 刘淑杰；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2002， 硕士

【摘要】 前言 在牙齿发育期间，牙乳头间充质细胞分化为成牙本质细胞，形成原发性牙本质；在牙齿萌出后，无论年轻恒牙，还是成熟恒牙，受损伤后均有修复性牙本质的形成，表明牙髓中存在有分化潜力的祖细胞群(progenitor cells)，后期可能分化为成牙本质细胞样细胞(odontoblast-like cells)。目前认为一些生长因子可能参与调节牙髓组织中的细胞增殖、分化和基质合成。碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)是一种多肽调节因子，对绝大多数来自中胚层和神经外胚层的细胞均具有刺激增殖活性，在胚胎形成、血管新生、神经营养及创伤愈合等方面起重要作用。迄今国内外学者对bFGF在人、鼠牙胚发育过程中的表达进行了免疫组化研究，认为bFGF与成釉细胞、成牙本质细胞的分化成熟及牙本质基质的沉积有关。本实验首次运用SP免疫组化技术和图像分析手段，显示bFGF在恒牙牙根不同发育阶段的牙髓中的表达及分布特征，探寻其在牙髓发育成熟中的作用。 材料与方法 牙髓标本取自多生牙、因阻生而拔除的第三磨牙或因正畸而拔除的健康无龋牙齿。所有研究对象均无全身系统性疾病，近三个月未曾服用免疫制剂和抗菌药物。将研究对象分成三组，即牙根刚开始发育(第一组)、牙根发育中(第二组)、牙根发育完成(第三组)。牙齿拔除后立即将牙齿纵向劈开，完整取出牙髓组织，放入4％的多聚甲醛液中，4℃下固定。梯度酒精脱水，二甲苯透明，石蜡包埋。每个标本于牙髓组织纵向最大面积处连续切片，厚5卜m，做 SP免疫组化染色。光学显微镜下观察阳性染色的分布及强度。 利用图像分析仪和Meta Morph／Cool snap仇AX70软件系统对阳性染色部位的透光强度进行定量分析问灰度值的测定人随机选取8个视野，对每张切片的冠髓与根髓组织的外层部分和中心区分别进行测量，结果取其平均值。用SPSS软件做统计学分析。 实验结 果 在牙髓组织中，bFGF主要在成纤维细胞、成牙本质细胞及血管壁内皮细胞的胞浆中呈阳性着色，同时在细胞外基质中呈线网状分布。年轻恒牙第一组牙髓的bFGF呈强阳性表达，第H组次之，第三组成熟恒牙牙髓的bFGF呈弱阳性表达。随着牙根的发育成熟，牙髓中阳性表达的成纤维细胞数逐渐减少。第一组牙髓外层的胜CF灰度值大于中心区（P＜O．001八第二组冠髓中心区的m＊F灰度值大于冠髓外层评＜0．05八成熟恒牙的冠髓外层灰度值大于年轻恒牙河＜O．001人各实验组的冠髓中心区灰度值两两之间比较，有非常显著性差异泽＜O．00八年轻恒牙的根髓中心区灰度值低于外层（P＜0．05厂且低于成熟恒牙的根髓中心区灰度值（P＜O．00）。 讨 论 国内外学者对牙髓细胞的体外培养实验研究表明，bFGF能促进牙髓细胞DNA的合成，是牙髓细胞的潜在有丝分裂原；加人bFGF使牙髓细胞的碱性磷酸酶（Alkaline PhosPhatase，ALPase）合成受到抑制，而Aryase诱导牙髓细胞的终末分化和钙化。成牙本 ·2·质细胞为终末细胞，失去增殖活性，bFGF对牙髓细胞的终末分化和钙化的抑制作用对于牙髓发育和修复中牙髓细胞的募集（re－cmiboent）是必备的先决条件。 近年来的研究表明bFGF参与牙胚的形态发生和牙齿形成细胞的分化。本实验采用免疫组化技术，发现牙髓中的成纤维细胞表达bFGF，而且随着牙根的发育，牙髓中表达bFGF的成纤维细胞数目逐渐减少，其染色强度逐渐减弱。因此，我们认为bFGF对牙髓细胞的发育和成熟起着重要作用。实验结果显示年轻恒牙牙根刚开始发育时，牙髓中心区的bFGF阳性表达强于牙髓外层，这可能是造成年轻恒牙牙髓中心区ALPase活力减弱，髓腔壁和壁附近的成牙本质细胞中ALPase活力最强的原因之一。有学者认为，牙髓细胞在分化为成牙本质细胞之前，需要经过一个去分化的过程，即ALPase活力下降，细胞进人分化潜力大的中胚层细胞，进而酶活力升高，分化为成牙本质细胞。因为ALPase的活性与牙髓的形成功能密切相关，所以bFGF可能通过控制ALPase的活性而参与了牙髓细胞的去分化过程，从而调节牙髓细胞向成牙本质细胞的分化。bFGF的阳性表达在年轻恒牙的冠髓外层强于冠髓中心区，根髓中心区强于根髓外层。由此我们推测年轻恒牙冠髓的成牙本质细胞附近的牙髓细胞以及根髓中心的牙髓细胞具有较强地分化为成牙本质细胞的能力。bFGF表达于牙髓细胞外基质kx－racellular matriX，ECM）中，表明成纤维细胞分泌的 bFGF可能被贮存在ECM中，从而刺激成纤维细胞的生长和分化。细胞的分化是由牙髓ECM来调节的。bFGF能与IV型胶原，层粘连蛋白（laminin，LN）及纤维粘连蛋白（ibronechn，FN）等ECM结合，这可以用来解释牙本质基质沉积过程中成牙本质细胞层和牙髓ECM中bFGF的强染现象，同11寸揭示了 bFGF在与ECM成分的相互作用以及伴随牙根发育的牙本质沉积中的重要作用。概括而言，bFGF释放后，一部分与周围细胞或自身胞膜受体结合，促进更多还原

【Abstract】 PrefaceDental papilla mesenchymal cells differentiate into primary odon-toblasts and form primary dentin during tooth development. After e-ruption, a certain fraction of pulp cells differentiate into odontoblasts and form secondary dentin. Investigations suggested that some growth factors might modulate proliferation and matrix synthesis in pulp. However the precise mechanisms remain unknown. Scholars have described the spatial - temporal expression of basic fibroblast growth factor (bFGF) during odontogenesis of mouse and human embryo as revealed by immunohistology. They believed that bFGF was involved in the odontoblast differentiation and dentin matrix deposition. But study on the expression and distribution of bFGF in pulps of permanent teeth during root development has not been reported. The purpose of this study was to discuss the significance of bFGF in the development and maturation of dental pulp.For the first time, by use of immunohistochemical and image a-nalysis techniques, we presented the expression and distribution pattern of bFGF in pulps of permanent teeth at different root development stage.Materials and MethodsDental pulps were obtained from healthy caries - free permanent teeth in need of extraction. All subjects had no systematic disease and didn’ t take any immune - inhibited drugs within three months. Based on the root development status, the pulp tissue was classified into three groups; root just starting development, root being in development and root finishing development. After extracted, the dental pulp was rapidly removed, then fixed in 0. 4% paraformaladehyde ( pH 7.4) at4C for 48 hours. Tissue blocks were dehydrated in ascending series of ethanol, cleared in xylene and embedded in paraffin. Serial sections were cut 5um thick. Anti - human poly clone antibody was used as the primary antibody and SP immunohistochemistry method was used for staining. The samples were observed under light microscope. Meta Morph/cool snapfx/AX70 image analysis software was used to detect the gray value of bFGF positive stained in dental pulp. The data were statistically analyzed by use of SPSS.ResultsStaining of immunohistochemistry for bFGF in dental pulps revealed that there were positive reactions in the cytoplasm of fibroblast, odontoblast, and epithelial cells in blood vessel wall and in extracellular matrix as well. Staining was strongly positive in immature permanent teeth, especially at the stage of root just starting development. The number of fibroblasts in positive expression was the most in them, and the least in the mature permanent teeth.Image analysis indicated that with the development of root formation , the gray value of the positive reaction in three groups were higher and higher and were significantly different ( P <0. 001) . For the first group, the gray value of the outer part was higher than that of the pulp core. For the second group, the pulp core has a higher gray value in the coronal pulp, while a lower value in root pulp compared to the outer parts. For the third group, there was no statistical difference between gray values in different pairts.DiscussionStudies of pulp cells in cultures indicated that adding bFGF enhanced DNA synthesis, whereas decreased ALPase level. ALPase may induce the terminal differentiation and calcification of pulp cells. Because terminal cells lose proliferate activity, the inhibition of terminal differentiation by bFGF may be a prerequisite for the recruitment of pulp cells during tooth development and repair.Recent investigations suggested that bFGF might be involved in tooth morphogenesis and differentiation of tooth - forming cells. By immunohistology we observed bFGF staining in pulp fibroblasts. With development of the root, the cell number expressed bFGF becomes less and the staining intensity weaker. It appears that bFGF may play a role in the development and maturation of pulp cells. The results indicate that when the young permanent teeth initially develop, bFGF positive expression in pulp core is strong更多还原