大鼠牙胚发育过程中釉原蛋白的表达

目的：研究釉原蛋白在大鼠牙胚发育不同时期的表达。方法;免疫组织化学和病理学方法。结果：胚胎第17天未观察到釉原蛋白的表达。第18天最先观察到免疫染色。出生后第3天和第5天成釉细胞分泌期釉基质中染色最强烈,以后处于低水平持续状态（第6、7、8天）。第9天染色为阴性。结论：釉原蛋白只在前成釉细胞处于分泌阶段和成釉细胞正分泌基质的时候才出现,在分泌后期,釉原蛋白的表达处于一种很低的水平。一旦分泌完成或釉质已形成,釉原蛋白就降解消失,在成牙本质细胞中未检测到釉原蛋白的表达。     

釉蛋白在大鼠牙胚发育过程中的转录表达

目的　观察釉蛋白在大鼠牙胚发育过程中的转录表达 ,为进一步研究釉蛋白的生物学功能提供基础。方法　采用原位杂交方法 ,检测出生后 1、3、7、10、14d龄大鼠第一磨牙牙胚中釉蛋白mRNA的表达。结果　大鼠出生后 1～ 10d在成釉细胞和成牙本质细胞中均检测到釉蛋白mRNA的表达。在成釉细胞中的表达随细胞的分化、基质分泌具有规律性 ,1d已有微弱表达 ,3d表达增强 ,7d达到最强 ,10d减弱 ,14d为阴性。釉蛋白mRNA在成牙本质细胞中的表达 1d很微弱 ,14d阴性 ,3～ 10d为持续的中等强度的表达。结论　釉蛋白在成釉细胞中的转录表达从前成釉细胞一直持续到成熟期 ,至牙冠硬组织发育完全时中止。成牙本质细胞也表达釉蛋白基因 ,提示釉蛋白可能参与早期罩牙本质的形成     

釉原蛋白在大鼠牙胚发育过程中的表达

目的 研究大鼠切牙牙胚形态分化的不同阶段,釉原蛋白(AmelogeninAm)的表达,探讨釉器细胞的形态分化与功能状态的关系,为探明牙釉质与牙本质在发育、矿化过程中的相互诱导关系及牙釉质蛋白硬组织诱导能力的有无提供实验依据。方法 将标本进行GMA树脂包埋,制作半薄切片后,进行免疫组织化学染色。结果 大鼠牙胚基底部釉器无Am的表达,分化末期内釉上皮细胞呈弱阳性染色,釉基质形成期成釉细胞及釉基质内呈强阳性染色。达到釉基质移行期后阳性反应逐渐减弱,直到釉质成熟期后期阳性反应消失。同时在对应的牙本质和牙本质小管内可以见到釉原蛋白的阳性染色。结论 在釉质形成过程中,成釉细胞分泌的釉原蛋白大部分存留在釉基质内形成釉质,也有一部分向牙本质内扩散到牙本质、牙本质小管、以及成牙本质细胞层内。釉原蛋白向牙本质层扩散和渗透功能上的意义,可能是起到促进成牙本质细胞的分化和诱导牙本质钙化的作用。     

釉原蛋白在大鼠胚发育过程中的表达

目的 研究大鼠切牙牙胚形态分化的不同阶段,釉原蛋白（Amelogenin Am）的表达,探讨釉器细胞的形态分化与功能状态的关系,为探明牙釉质与牙本质在发育、矿化过程中的相互诱导关系及牙釉质蛋白硬组织诱导能力的有无提供实验依据。方法 将标本进行GMA树脂包埋,制作半薄切片后,进行免疫组织化学染色。结果 大鼠牙胚基底部釉器无Am的表达,分化末期内釉上皮细胞呈弱阳性染色,釉基质形成期成釉细胞及釉基质内呈强阳性染色。达到釉基质移行期后阳性反应逐渐减弱,直到釉质成熟期后期阳性反应消失。同时在对应的牙本质和牙本质小管内可以见到釉原蛋白的阳性染色。结论 在釉质形成过程中,成釉细胞分泌的釉原蛋白大部分存留在釉基质内形成釉质,也有一部分向牙本质内扩散到牙本质、牙本质小管、以及成牙本质细胞层内。釉原蛋白向牙本质层扩散和渗透功能上的意义,可以是起到促进成牙本质细胞的分化和诱导牙本质钙化的作用。     

成釉蛋白在小鼠牙胚发育不同时期的免疫组化定位

目的：观察成釉蛋白（ameloblastin,AMBN)在小鼠牙胚发育不同时期的表达和分布情况。方法：采用免疫组化的方法观察AMBN在小鼠牙胚不同时期的表达和定位。结果：蕾状期、帽状期和钟状早期牙胚中未见AMBN的表达；出生后1d钟状期，在成釉细胞和釉基质中呈强阳性表达，成牙本质细胞和牙本质基质中呈弱阳性表达；出生后3d，在成釉细胞胞浆中呈弱阳性表达，在成牙本质细胞中表达阳性，着色较成釉细胞深；出生后7d，AMBN在成釉细胞托姆氏突呈弱阳性表达，其余部位呈阴性表达。在牙胚发育的各个时期，AMBN在外釉细胞、星网层细胞、牙乳头细胞均呈阴性表达。结论：AMBN参与釉基质的形成，可能与牙胚发育中基质形成的信息传递有关。     

原癌基因c-jun、c-fos在大鼠牙胚发育过程中的表达

目的：通过观察原癌基因c—jun、c—fos在大鼠牙胚发育过程中时空表达的特点，探讨它在牙胚发育中的作用。方法：制备大鼠牙胚发育各期组织标本，采用免疫组化的方法观察c-jun、c-fos在大鼠牙胚发育不同阶段的表达情况，并对结果进行图像分析和统计学分析。结果：c-jun在大鼠牙胚的蕾状期、帽状期呈阴性表达，在钟状晚期牙胚的成牙本质细胞中呈阳性表达，成釉细胞中亦有表达；c-fos在大鼠牙胚的蕾状期呈阴性表达，帽状期表达于内釉、外釉上皮和星网层，以内釉上皮层表达最强。钟状期表达于成熟的成牙本质细胞，且随着硬组织的形成、矿化，表达逐渐减弱。结论：观察到c-jun、c-fos在大鼠牙胚发育不同时期的表达和表达变化，提示其可能参与成牙本质细胞、成釉细胞的分化和牙本质形成的过程。     

Notch 1信号蛋白在大鼠牙胚发育中的免疫组化表达研究

目的通过检测大鼠牙胚发育不同阶段Notch1信号的表达情况，探讨其在牙齿发育过程中的作用。方法采用免疫组织化学染色技术观测大鼠孕期12、14、16、17、18、19d和出生后1、3、5、7、9d牙胚中Notch1的表达。结果Notch1的表达始于钟状中期。在钟状中晚期，Notch1的表达在牙乳头间充质、成牙本质细胞层呈弱阳性。在内釉细胞层呈阳性。牙体组织形成开始，Notch1则在成釉细胞的中间层有反复表达，在成牙本质细胞层中有一过性表达。牙齿萌出后，该信号在成牙本质细胞和成釉细胞表达均为阴性。结论Notch1在牙胚发育过程中与成釉细胞分化有关，它可能在牙齿发育早期基质触发前成釉细胞向成釉细胞分化中起重要作用。     

小鼠牙胚发育过程中釉原蛋白和釉蛋白的表达特征

目的 观察釉原蛋白和釉蛋白在小鼠牙胚发育中的表达特征,进一步揭示釉原蛋白和釉蛋白的生物学特性.方法 分别制备出生后第1、3、7和14天小鼠下颌第一磨牙牙胚切片,采用免疫组织化学和反转录聚合酶链反应法检测釉原蛋白和釉蛋白的组织学定位和基因转录表达水平.结果 釉原蛋白表达于分泌期成釉细胞的胞质和釉质基质全层,在新生小鼠牙胚成牙本质细胞中有一过性表达;釉蛋白表达于分泌期釉质基质中,在成釉质细胞突边缘和釉质基质深层呈强阳性表达,釉原蛋白和釉蛋白在矿化成熟的釉质中均未见表达.釉原蛋白和釉蛋白在出生后第7天mRNA表达量的相对值分别为0.813±0.085和0.799±0.064,显著高于其他时间的表达水平(P＜0.05).结论 釉原蛋白和釉蛋白主要由分泌期成釉细胞合成并分泌到釉质基质中,其表达具有高度的时空特异性,提示它们在釉质形成和生物矿化中有重要的作用.     

釉原蛋白在人牙胚发育不同时期表达的免疫组化研究

目的：探讨釉原蛋白在人牙胚发育不同时期的表达及其意义。方法：采用免疫组织化学方法观察釉原蛋白在不同发育阶段人牙胚中的分布。结果：在前成釉细胞、基底膜及前成牙本质细胞相邻部位都呈阳性反应。牙乳头细胞及牙本质呈阴性反应。釉原蛋白分布于釉质全层，在釉质浅层及与成釉细胞界面处呈线状强阳性染色，在后期矿化成熟时，成釉细胞及釉质染色阴性。结论：釉原蛋白与成釉细胞、成牙本质细胞的分化及釉质的矿化有关。     

核心结合因子a1在小鼠牙齿发育过程中的表达

目的：观察Cbfal蛋白在小鼠牙胚发育过程中的表达情况。方法：用自制的Cbfal多克隆抗体，采用免疫组化染色观察其时空表达特性。结果：Cbfal蛋白在帽状期牙胚中表达较广泛，在钟状早、中期牙胚表达于内釉细胞、成釉细胞以及紧靠成牙本质细胞层的牙乳头细胞，而在钟状晚期，Cbfal在牙乳头表达为阴性，成牙本质细胞层弱阳性，成釉细胞为阳性或强阳性。结论：Cbfal可能在小鼠牙胚发育，尤其是在成牙本质细胞和成釉细胞分化过程中具有重要作用。     

Ameloblastin and amelogenin expression in posnatal developing mouse molars

Ameloblastin and amelogenin are structural proteins present in the enamel matrix of developing teeth. Here we report the results of in situ hybridization analyses with DNA probes of ameloblastin and amelogenin expression in the mandibular first molars of ICR/Jcl mice from postnatal day 1 to day 15. Ameloblastin mRNA expression was observed in ameloblasts at day 2 while amelogenin mRNA was detected in secretory ameloblasts at day 3. Significant expression of both molecules was observed at days 4, 5 and 6, after which the levels decreased. Amelogenin expression ended on day 10, while ameloblastin mRNA was only weakly detected on day 12. Neither amelogenin nor ameloblastin expression was observed in day 15 mouse molars. Amelogenin and ameloblastin mRNAs were restricted to ameloblasts. We conclude that amelogenin and ameloblastin expression is enamel-specific, and suggest that these genes might be involved in the mineralization of enamel. It is possible that ameloblastin could participate in the attachment of ameloblasts to the enamel surface. In this case, the downregulation of expression may indicate the beginning of the maturation stage in which the ameloblasts tend to detach from the enamel layer.     

A comparison of enamelin and amelogenin expression in developing mouse molars

Amelogenin and enamelin are structural proteins in the enamel matrix of developing teeth. The temporal and spatial patterns of enamelin expression in developing mouse molars have not been characterized, while controversy remains with respect to amelogenin expression by odontoblasts and cementoblasts. Here we report the results of in situ hybridization analyses of amelogenin and enamelin expression in mouse molars from postnatal days 1, 2, 3, 7, 9, 14, and 21. Amelogenin and enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onsets of amelogenin and enamelin expression were approximately synchronous with the initial accumulation of predentin matrix. Both proteins were expressed by ameloblasts throughout the secretory, transition, and early maturation stages. Enamelin expression terminated in maturation stage ameloblasts on day 9, while amelogenin expression is still detected in maturation stage ameloblasts on day 14. No amelogenin expression was observed in day 21 mouse molars. Amelogenin and enamelin RNA messages were restricted to ameloblasts. No expression was observed in pulp, bone, or along the developing root. We conclude that amelogenin and enamelin are enamel-specific and do not directly participate in the formation of dentin or cementum in developing mouse molars.     

通过真核细胞表达系统获得重组人釉原蛋白

目的构建重组人釉原蛋白基因的真核表达系统，并建立稳定表达该蛋白的细胞系。方法取26周龄引产胎儿的牙胚组织，提取总RNA，用RT—PCR技术扩增釉原蛋白基因片段，插入中间表达载体pGEM -T。经双酶切后，再与真核表达载体pcDNA3.1^TM／myc—His（-）B相连接，构成最终的表达质粒，将该重组表达质粒转染至HEK 293A细胞，用G418筛选出阳性细胞克隆，并建立稳定表达釉原蛋白的细胞系。结果通过测序表明，人釉原蛋白基因被成功地连接到了真核表达载体上。将该表达系统转染HEK293A细胞后，进行Western Blot检测，证明有相对分子质量约32000的釉原蛋白表达。结论本实验成功构建了重组人釉原蛋白真核表达系统，建立了稳定细胞系，为获得高纯度的釉蛋白，进一步研究蛋白质功能奠定了基础。     

大鼠釉原蛋白基因表达的载体构建及其在大肠杆菌中的表达

目的 观察釉原蛋白（ａｍｅｌｏｇｅｎｉｎ,Ａｍ）基因聚合酶链反应（ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ,ＰＣＲ）产物在大肠杆菌中的表达。方法 利用谷胱苷肽巯基转移酶表达载体４Ｔ－２型（ｐｔａｃ ｇｌｕｔａｈｉｏｎ ｅｘｐｒｅｓｓｉｏｎ,ＰＧＥＸ－４Ｔ－２）载化大肠杆菌ＤＨ５α,通过异丙基硫代半乳糖苷（ｉｓｏｐｒｏｐｙｌｔｈｉｏ－ｇａｌａｃｔｏｓｉｄｅ,ＩＰＴＧ）诱导,收菌。聚丙烯酰胺凝胶电泳。结果 电泳分析表明,釉原蛋白基因ＰＣＲ产物在大肠杆菌中成功地获得了表达。结论 构建了ＰＧＥＸ－４Ｔ－２／Ａｍ融合表达载体,获得了融合表达的目的蛋白。     

Gene expression and immunolocalization of amelogenin in enamel hypoplasia induced by successive injections of bisphosphonate in rat incisors

Successive injections of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) in rats induce enamel hypoplasia. To elucidate the pathogenesis of this hypoplasia, male Wistar rats were daily injected with HEBP or physiological saline for 7 days. After the last injection, they were killed under anaesthesia and their maxillary incisors were examined using an in situ hybridization technique and immunohistochemical staining to detect the gene expression and localization of amelogenin protein, respectively. In the HEBP-injected rats, several islets of partially mineralized enamel were present along crown-analogous surface of the incisor in the secretory stage of amelogenesis and enamel-free zones existed between these islets. In situ hybridization demonstrated amelogenin gene expression over the ameloblasts facing the islets of the matrix enamel as well as over those of the enamel-free zones. Immunohistochemical studies using rabbit antiamelogenin antibody revealed positive reaction both in the enamel matrix of the control group and in the islets of enamel matrix of the HEBP-injected group. Some small granules immunoreactive to amelogenin antibody were found in the distal portions of the ameloblasts in the HEBP-injected rats. The results indicate that HEBP does not alter amelogenin gene expression over ameloblasts, or the protein's existence in enamel matrix. There appeared to be some accumulation of amelogenin in the HEBP-treated ameloblasts. It is therefore suggested that the enamel hypoplasia in this experiment may not be due to a disturbance in amelogenin synthesis but to a disturbance in a later process, presumably of protein secretion.     

Phex mutation causes overexpression of FGF23 in teeth

OBJECTIVE: Hyp mice have a disorder in phosphate homeostasis, and display hypo-mineralization in bones and teeth, while the Phex (phosphate regulating gene homologies to endopeptidase on the X chromosome) gene in Hyp mice has a deletion of the 3' end. We investigated whether a mutation of Phex has an effect on the expression level of fibroblast growth factor 23 (FGF23), one of the key factors of phosphate homeostasis, in developing teeth of Hyp mice. DESIGN: RT-PCR and in situ hybridisation analyses for FGF23 were performed using developing teeth of WT mice. Quantitative RT-PCR analyses for FGF23 were performed using the tooth germs of WT and Hyp mice in both in vivo and in vitro experiments. RESULTS: Undifferentiated and early secretory ameloblasts as well as odontoblasts expressed FGF23 mRNA during early tooth development. Further, quantitative RT-PCR analyses revealed that the amount of FGF23 mRNA in Hyp mouse teeth was significantly higher than that in wild type mice. CONCLUSIONS: These findings suggest that loss of Phex function is related to overexpression of FGF23 in teeth, which is an intrinsic defect of Hyp mouse teeth.     

Enamel Formation —Biochemical Aspect—

This review shows the biochemical aspect of enamel formation focused on porcine amelogenesis. Volumetric changes in the chemical components of secretory stage enamel intimate that enamel crystals thicken as amelogenin, the major protein component, decreases. Solubility behaviors and hydrophobicity analyses of amelogenin derivatives suggest the hypothesis that cylindrical amelogenin forms micelles. This model explains how amelogenin constructs a large structure, which forms a matrix, and continuously yields space as the crystals thicken. In porcine secretory enamel, mineral volume increases with depth, gaining space for thickening the existing crystallites by shrinking amelogenin micelles through the removal of Cterminal and 13-kDa peptides from the prototype 25-kDa amelogenin, the most abundant porcine amelogenin gene product, by the action of enamelysin (MMP-20) and enamel matrix serine proteinase (EMSP1), respectively.During the secretory stage, the crystallites thicken in the deeper secretory enamel, although calcium ions are supplied by ameloblasts. Another morphological feature of the crystallites is that they are all nearly the same size within the same developmental stage. There are several enamel proteins and their cleavage products have calcium binding activity and binding affinity for crystallites. The 25-kDa amelogenin and 89-kDa enamelin, both neutral insoluble, are involved in slow crystallite growth by trapping calcium ions secreted by ameloblasts, and may control by inhibiting newly nucleated crystals between existing crystallites. The calcium binding proteins derived from the C-terminal side of sheathlin (ameloblastin/amelin) and 32-kDa enamelin, belonging to the neutral soluble fraction, act as a calcium carrier to transport calcium ions to deeper secretory stage enamel. Sheath protein derived from the N-terminal side of sheathlin is the main protein constituent of the enamel sheath.Enamelin may be involved in initial crystal formation as it is immuno-histochemically shown to be localized on or around the initial nucleated crystals in a presecretory stage matrix. This is confirmed by no crystal formation in the enamel of enamelin knock-out mice. This conflicts with the inhibition of new nucleation between the existing crystallites in advanced secretory stage enamel. Enamelin may have multi-functions which are involved in the induction of nucleation in a particular phase of the molecule and inhibition in other phases, or change its function with the products produced during each degradation step.     

Evolution of the amelogenin gene in toothed and toothless vertebrates

Amelogenin proteins constitute the major organic contents in forming enamel, but some previously published data indicate that the amelogenin gene could be present in several species lacking teeth or functional enamel. Therefore, amelogenin could have another, still unknown, function other than contributing to enamel formation. In order to test this hypothesis, the presence of this gene has been searched for in various vertebrates. We first compared the mammalian amelogenin sequences available in the literature to obtain the best chance of a successful detection of this gene in phylogenetically distant species. Using this analysis, we have shown that the occurrence of the amelogenin gene in the Y chromosome of primates and of an artiodactyl species is probably due to two independent duplications from genes on the X chromosome. Primers for PCR have therefore been synthesized and tested in eight species, five possessing teeth (human, two phylogenetically distant lizards, and two phylogenetically distant actinopterygians) and three lacking teeth (chicken and two phylogenetically distant turtles). The amelogenin gene has been detected in all species except those lacking teeth. This result indicates that the unique role of amelogenin in amniotes is to contribute to enamel formation.