人釉原蛋白全长及其功能片段的体外自组装和矿化行为的研究

目的 探讨人釉原蛋白(AM)全长及其N端酪氨酸富集段(TRAP)、C端亮氨酸富集段(LRAP)体外自组装的动态过程及其在羟磷灰石(HA)晶体形成中的作用.方法 体外重组、纯化人AM全长及其功能片段TRAP、LRAP,在三氨基甲烷(Tris-HCl)中配制成100μg·mL-1、pH=8的蛋白溶液,室温孵育1～15 mi...     

Dentine structure and mineralization in hypocalcified amelogenesis imperfecta: a quantitative X-ray histochemical study

OBJECTIVE: This study was undertaken in order to establish the structural and mineralization pattern of the response of dentine to alterations in enamel in hypocalcified amelogenesis imperfecta (AI). DESIGN: The images and data obtained with scanning electron microscopy and electron probe X-ray microanalysis in enamel and dentine specimens from control and affected teeth were compared in this study. PATIENTS AND METHODS: We compared 46 fragments of permanent teeth from patients with clinically diagnosed hypocalcified AI and 20 normal permanent teeth. All specimens were prepared for electron probe X-ray microanalysis. RESULTS: Dentine is characterized by thickening of the peritubular dentine and partial obliteration of the dentinal tubules that does not give rise to a compact sclerotic cast. In dentine, calcium levels were significantly higher in teeth with clinically hypocalcified AI in relation with control teeth (P < 0.001). CONCLUSIONS: Dentine is affected in hypocalcified AI increasing mineralization (narrower tubules and higher content of calcium) in response to enamel disorder.     

Effect of calcium, given before or after a fluoride insult, on hamster secretory amelogenesis in vitro

We tested the hypothesis that high-calcium medium given prior to or immediately after exposure to fluoride (F) reduces the negative effects of F on secretory amelogenesis. Hamster molar tooth germs were grown in organ culture in media with different calcium levels. Deposition of enamel matrix and matrix mineralization were monitored by incorporation of [³H]proline and uptake of ⁴⁵Ca and acid-soluble ³²PO₄. Ameloblast structure and the occurrence of a fluorotic enamel matrix were examined by light and electron microscopy. A preculture of explants in high-calcium medium partially prevented the formation of fluorotic (non-mineralizing) enamel matrix, increased matrix secretion but could not prevent F-induced hypermineralization of the pre-exposure enamel. High-calcium medium, applied after F insult, accelerated the recovery of fluorotic matrix, improved ameloblast structure, enhanced amelogenin secretion, and increased enamel thickness. The data indicate that it might be the balance between the amount of mineral deposition and that of matrix secretion which is critical for the mineralization of newly secreted enamel. Exposure to F disturbs this balance by enhancing mineralization of the pre-exposure enamel, probably generating an excess of protons. High calcium may protect against F exposure by enhancing amelogenin secretion into the enamel space, thereby increasing the local buffering capacity at the mineralization front.     

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。     

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.     

胞内信号转导分子LIM矿化蛋白-1在人牙周膜细胞体外矿化过程中表达的实验研究

目的:研究胞内信号转导分子LIM矿化蛋白-1在人牙周膜细胞体外矿化过程中的表达。方法:利用组织块法体外培养人牙周膜细胞,实验组培养液中加入矿化诱导剂,对照组不加矿化诱导剂。培养14 d后,免疫细胞化学检测骨涎蛋白的表达;von Kossa染色检测矿化结节的形成。培养期间,采用半定量RT-PCR技术监测LIM矿化蛋白-1、碱性磷酸酶的表达,SPSS11.0软件分析结果。结果:培养14 d,实验组骨涎蛋白免疫细胞化学呈阳性表达,von Kossa染色阳性,矿化结节形成;对照组骨涎蛋白表达阴性,von Kossa染色阴性。RT-PCR结果显示碱性磷酸酶、LIM矿化蛋白-1在实验组和对照组各时段均表达。培养期间,实验组LIM矿化蛋白-1的表达显著高于对照组,其中培养第3 d、14 d时表达分别达到两个高峰,约为对照组的1.4倍、1.5倍。培养期间,碱性磷酸酶的表达显著增高,其中培养14 d时约为对照组的1.5倍。结论:首次发现LIM矿化蛋白-1与人牙周膜细胞的体外矿化过程相关,提示LIM矿化蛋白-1可能在牙周膜细胞的分化、矿化中发挥一定作用。     

Human ameloblastin gene: genomic organization and mutation analysis in amelogenesis imperfecta patients

A gene encoding the enamel protein ameloblastin (AMBN) was recently localized to a region on chromosome 4q21 containing a gene for the inherited enamel defect local hypoplastic amelogenesis imperfecta (AIH2). Ameloblastin protein is located at the Tomes processes of secretory ameloblasts and in the sheath space between rod-interrod enamel, and the AMBN gene therefore represents a viable candidate gene for local hypoplastic amelogenesis imperfecta (AI). In this study, the genomic organization of human AMBN was characterized. The gene was shown to consist of 13 exons and 12 introns. An alternatively spliced 45 bp sequence was shown not to represent a separate exon and is most likely spliced by the use of a cryptic splice site. The finding that there were no recombinations between an intragenic microsatellite and AIH2 encouraged us to evaluate this gene's potential role as a candidate gene for local hypoplastic AI. Mutation screening was performed on all 13 exons in 20 families and 8 sporadic cases with 6 different forms of AI. DNA variants were found but none that was associated exclusively with local hypoplastic AI or any of the other variants of AI in the identified Swedish families. This study excludes the coding regions and the splice sites of AMBN from a causative role in the pathogenesis of AIH2.     

体外磷灰石晶体矿化模型构建及应用的初步研究

目的构建磷灰石晶体矿化的体外模型,研究牛血清白蛋白（BSA）和不同浓度的氟离子对磷灰石晶体体外矿化的影响。方法利用阳离子膜和透析膜建立磷灰石晶体矿化的体外模型。在模型中分别加入去离子水、BSA和不同浓度的氟离子（5、20、100 mg·L-1）,反应3 d后,扫描电镜（SEM）、X线衍射法检测矿化产物。结果成功构建了磷灰石晶体体外矿化模型,加入去离子水和BSA后,晶体成分主要为磷酸八钙（OCP）,但晶体形态差异显著。加入氟离子后,形成了柱状、杆状的氟磷灰石（FAP）晶体,且随着氟离子浓度的增加,晶体的尺寸和矿化程度均增加。结论该体外模型是检测磷灰石晶体矿化调控因素的有效方法。     

永生化人成牙本质细胞样细胞系体外矿化的初步观察

目的:探讨永生化人成牙本质细胞样细胞系hTERT-hOd-1的体外矿化特征。方法：在矿化液连续培养细胞5周，采用倒置显微镜、透射电镜进行组织学观察以及Von Kossa染色。结果：细胞在矿化液中生长良好，形成细胞结节，分泌细胞外基质，其中有平行排列的胶原纤维和针状晶体沉积。Von Kossa染色显示细胞结节已发生矿化。结论:hTERT-hOd-1细胞在体外培养条件下具有矿化能力。     

重组人釉原蛋白促进人脐静脉内皮细胞成血管分化作用的研究

目的 观察重组人釉原蛋白(recombinant human amelogenin, rhAm)对体外培养的人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC)成血管作用的影响。方法 体外培养HUVEC,分别将浓度为0.1、10.0、50.0、100.0μg/mL的rhAm作用于细胞,与对照组比较,采用MTT法观察HUVEC增殖,通过划痕实验观察HUVEC迁移,运用实时定量PCR和免疫印迹检测rhAm对HUVEC表达成血管相关基因及膜蛋白的影响,经由体外小管生成实验观察HUVEC小管样结构。结果 MTT结果显示,0.1μg/mL和10.0μg/mL rhAm可促进HUVEC的增殖(P<0.05),100.0μg/mL rhAm出现抑制HUVEC增殖的现象(P<0.01);划痕实验结果显示,10.0μg/mL rhAm可促进HUVEC迁移(P<0.05);实时定量PCR结果显示,10.0μg/mL rhAm可上调细胞间黏附分子-1(intercellular cell adhesion molecule-1,ICA...     

FHL2蛋白在人牙周膜细胞体外矿化过程中的表达

目的 探讨胞内信号转导分子FHL2蛋白在人牙周膜细胞(hPDLCs)体外矿化过程中的表达.方法 体外培养hPDLCs,实验组用矿化诱导液培养,对照组不加诱导液.培养0、14、28 d后,茜素红染色检测矿化结节的形成;免疫细胞化学法检测hPDLcs矿化诱导0、14d时FHL2蛋白的表达;同时采用半定量RT-PCR方法检测...     

Identification of a novel FAM83H mutation and microhardness of an affected molar in autosomal dominant hypocalcified amelogenesis imperfecta

Aim  To determine the underlying molecular genetic aetiology of a family with the hypocalcified form of amelogenesis imperfecta and to investigate the hardness of the enamel and dentine of a known FAM83H mutation.
Methodology  Mutational screening of the FAM83H on the basis of candidate gene approach was performed. All exons and exon–intron boundaries was amplified and sequenced. A microhardness test was performed to measure the Vickers microhardness value.
Results  A novel nonsense mutation (c.1354C>T, p.Q452X) was identified in the last exon of FAM83H , which resulted in soft, uncalcified enamel. The affected enamel was extremely soft (about 17% of the normal control), but the underlying dentine was as hard as the normal control.
Conclusions  Mutational analysis revealed a novel mutation in FAM83H gene. Hardness of dentine was not affected by the mutation, whilst the enamel was extremely soft.     

Evaluation of oral and systemic manifestations in an amelogenesis imperfecta population

Objectives: The aim of this investigation was to describe the dental and craniofacial characteristics of patients with amelogenesis imperfecta (AI). Methods: The study group included 43 patients(33 female and 10 male) with a mean age of 11.4±2.6 years. A panoramic and a cephalometric radiograph were obtained from each of these patients. Clinically AI cases were divided into four main groups according to Witkop. All patients were evaluated for chronological, bone and dental age. The patients who had severe retarded bone age were evaluated for plasma growth hormone(GH) concentrations. Results: Dental and bone ages were retarded with respect to chronological age in five patients. Dental maturity and tooth eruption were not age- appropriate in some of our patients. In type III AI patients a delay in skelatal age was observed. Severe late eruption was seen in 3 patients, severe delay in dental maturity was noted in patients with type IV AI. Dental age was clinically lower in GH-deficient subjects, and skeletal age was consistently more retarded than dental age when compared to chronological age. Anterior open bite was present in both primary and permanent dentitions of 50% of the patients with type I AI, 30.8% of the patients with type II AI, and 60% of type III AI. Conclusion: It is concluded that the primary structure for the classification of AI be based on the mode of inheritance, with the clinical and radiographic appearances (and any other features such as systemic findings) being the secondary discriminators.     

cbfa1反义核酸对体外培养小鼠牙胚发育与矿化的影响

目的:采用反义核酸技术,用特异的与cbfa1mRNA互补的AS ODN抑制cbfa1在体外培养牙胚的翻译,利用电镜及ALP、OC等指标观察其对牙齿发育及矿化的影响,探讨其在牙齿发育中的作用。方法:设计合成cbfa1反义核酸,应用所建立的牙胚体外培养系统,用电镜观察小鼠牙胚被cbfa1反义核酸阻断表达后发育的情况,并且检测ALP、OC表达量的变化。结果:在缺乏cbfa1的情况下,与对照组相比,形成的基质较薄,且基质中胶原纤维排列稀疏,粗细不等,方向杂乱,ALP、OC表达量均有明显变化。结论:cbfa1可能是通过调控ALP和OC等靶基因的表达参与了牙齿发育矿化过程。     

硅和氟对人恒前磨牙釉质脱矿影响的体外研究

目的:评价硅、氟对离体恒前磨牙釉质脱矿的影响。方法:收集123个正畸拔除的恒前磨牙,制备釉质标本,随机分为对照组、1 mg/L硅(Si)组、1 mg/L氟(F)组,每组41个。将3组标本分别置去离子水、1 mg/L硅溶液、1 mg/L氟溶液预处理8 h、取出标本浸泡于酸蚀凝胶中,随后测定脱矿后7个时间点凝胶中的钙溶出量;并用扫描电镜观察24、168 h脱矿釉质表面超微结构变化。结果:不同脱矿时间,Si组钙溶出量与对照组相比无统计学差异(P﹥0.05);F组钙溶出量低于Si组和对照组,有统计学差异(P﹤0.05);扫描电镜观察各组釉质表面随脱矿时间的延长呈现进行性破坏现象,F组表面破坏程度最轻,168 h后3组蜂窝结构底部均见球状沉积物,Si组沉积物数目较F组多。结论:在体外条件下,1 mg/L Si溶液对前磨牙釉质无明显抑制脱矿作用,1 mg/L F溶液则可明显抑制釉质的脱矿。     

LIM矿化蛋白1在大鼠牙髓损伤修复中的时空表达

目的:探讨LIM矿化蛋白1(LIM mineralization protein 1,LMP-1)在大鼠磨牙牙髓损伤修复中的表达.方法:建立大鼠磨牙牙髓损伤修复动物模型,用免疫组织化学染色方法观察LMP-1在大鼠牙髓损伤修复中的表达,并用Image-Pro Plus 6.0图像分析软件及单因素方差分析比较各组间的差异.结果:在正常大鼠牙髓组织中LMP-1表达阴性;在大鼠牙髓损伤后1d,LMP-1表达于成牙本质细胞及部分牙髓成纤维细胞;术后3 d,LMP-1表达于坏死牙髓下方的细胞增殖层;术后7d,LMP-1显著表达于增殖活跃的牙髓细胞及部分成牙本质细胞中.结论:LMP-1在牙髓损伤修复过程中呈时空特异性表达,可能参与成牙本质细胞及牙髓细胞的增殖、分化及修复性牙本质的形成.     

Early in vivo and in vitro effects of amelogenin gene splice products on pulp cells

Recombinant amelogenin gene splice products A+4 and A-4, implanted in the pulp, induce the recruitment, proliferation, and differentiation of reparative cells. Our aim was to investigate the precocious events occurring in the pulp 1 d and 3 d after implantation of agarose beads alone or loaded with A+4 or A-4. Proliferation and cell recruitment towards an odonto/osteogenic phenotype were visualized by detection of the proliferation cell nuclear antigen (PCNA) and RP59. After implantation of beads alone or loaded with A+4, at day 3, pulp cells were moderately immunopositive for osteopontin (OP), whereas labeling was strongly positive upon treatment with A-4. Dentin sialoprotein (DSP) labeling was not detectable. Parallel in vitro studies were carried out on odontoblastic and mesenchymal progenitor cells in order to evaluate the effect of the amelogenin peptides on the expression of a series of marker genes involved in the odontoblastic/osteogenic/chondrogenic differentiation pathways. Altogether, our results suggest that the 'signaling' effects of the amelogenin peptides A+4 and A-4 may differ according to the type of target cells, their stage of differentiation, the time of treatment, and the type of amelogenin peptide (A+4 or A-4).