FAM20C在体外培养人牙髓细胞中的表达及成牙本质向分化诱导对其表达的影响

目的研究人牙髓细胞（hDPC）体外培养传代过程中FAM20C的表达模式,及对hDPC成牙本质向分化诱导后FAM20C的表达变化。 方法蛋白免疫印迹法（Western blot）检测第1代至第7代（P1 ~ P7）hDPC中FAM20C蛋白的表达量;结果采用独立样本t检验;对P3代hDPC进行成牙本质分化诱导7和14 d后,反转录聚合酶链反应与Western blot检测FAM20C的mRNA和蛋白水平变化。牙本质涎磷蛋白（DSPP）、牙本质基质蛋白1（DMP1）表达变化,FAM20C的mRNA以及蛋白表达变化采用单因素方差分析。免疫荧光法检测FAM20C在hDPC中的分布。 结果体外培养的hDPC中可检测到FAM20C表达,表达量随细胞传代先增加后减少（t_P2=-10.177,P_P2= 0.001;t_P3=-18.242,P_P3<0.001;t_P4=-19.143,P_P4<0.001;t_P5=-7.452,P_P5= 0.002;t_P6= 1.357,P_P6= 0.246;t_P7= 1.099,P_P7= 0.334）;成牙本质向分化诱导7和14 d后,FAM20C的mRNA和蛋白表达量高于未诱导组细胞（F_{FAM20C mRNA}=86.252,P_{FAM20C mRNA,7 d}<0.001,P_{FAM20C mRNA,14 d}<0.001;F_FAM20C蛋白= 85.569,P_{FAM20C蛋白,7 d}= 0.002,P_{FAM20C蛋白,14 d}<0.001）。免疫荧光结果显示,成牙本质诱导前FAM20C蛋白表达于细胞核,诱导后主要表达于细胞质,诱导14 d FAM20C在细胞质中表达量高于诱导7 d。 结论hDPC中表达FAM20C,成牙本质向分化诱导上调其表达水平,诱导后在细胞质中表达较高,提示FAM20C与hDPC的成牙本质分化相关。     

牙本质牙髓复合体的形成研究(二)牙本质牙髓复合体的在体形成研究

牙本质牙髓复合体在体形成研究包括牙齿发育完成后在各种因素下形成的第三期牙本质和成牙组织、细胞在口腔以外部位形成牙齿样结构两方面。一些细胞外基质分子和生长因子形成的网络调控在其形成中发挥关键性作用。对这些分子机理的研究将为临床牙髓病的生物保髓治疗和牙髓、牙齿再生替代治疗奠定了基础。     

bFGF在大鼠牙齿发育过程中表达的研究

目的:观察碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)在大鼠牙齿发育过程中的表达和分布变化,探讨bFGF在大鼠牙齿发育过程中的作用。方法:选用5、10、15、20、25d的大鼠,制备大鼠牙齿发育组织标本,采用免疫组织化学方法观察bFGF在大鼠牙齿不同阶段的表达情况。结果:在大鼠牙齿发育早期bFGF主要在成釉细胞表达,而在中间层和牙乳头表达较少。随着牙齿的发育,在成釉细胞、成牙本质细胞、前期牙本质和牙乳头表达逐渐加强。结论:bFGF可能与大鼠前期牙本质的沉积,牙釉质和牙本质的发育形成有关。     

牙本质磷蛋白与牙齿的生物矿化

牙本质磷蛋白是牙齿发育过程中由成牙本质细胞分泌的一类非胶原蛋白,是一组带负电荷的富酸性蛋白,在牙齿的矿化和再矿化中起重要作用。与人类遗传性疾病牙本质发育缺陷Ⅰ型密切相关。本文就近年来的研究状况进行综述。     

牙本质磷蛋白与牙齿的生物矿化

牙本质磷蛋白是牙齿发育过程中由成牙本质细胞分泌的一类非胶原蛋白,是一且带负电荷的富酸性蛋白,在牙齿的矿化和再矿化中起重要作用。与人类遗传性疾病牙本质发育缺陷Ⅱ型密切相关,本文就近年来的研究状况进行综述。     

牙本质牙髓复合体的形成研究(一)牙本质牙髓复合体的新概念及其在牙齿发育中的形成研究

牙本质及牙本质牙髓复合体的形成研究一直是口腔医学研究的重大课题。近年来,生命前沿科学领域的飞速发展和重大进展,也掀起了牙本质牙髓形成研究的热潮。本文阐述了对牙本质牙髓复合体概念的新认识,系统回顾了牙齿发育过程中、牙齿发育完成后,牙本质牙髓复合体体内形成研究、体外形成研究的新进展,及这些研究对临床治疗的指导意义和应对策略,分析了面临的挑战。这对我们把握该领域的研究现状和进一步深入研究奠定了必要的基础。     

牙本质磷蛋白mRNA原位杂交方法的建立

目的:建立检测牙本质磷蛋白(DPP)mRNA表达的原位杂交方法。方法:选用发育各阶段的牙胚、牙齿和体外培养的MDPC-23成牙本质细胞为对象,采用地高辛标记的寡核苷酸探针的原位杂交方法。结果:DPP mRNA在牙胚与牙齿中的成牙本质细胞、前成釉细胞和体外培养的成牙本质细胞存在阳性表达。结论:设计的探针敏感性高,特异性高,所建立的原位杂交方法是研究牙本质发育和损伤修复的良好方法。     

甲状旁腺激素对体外培养的大鼠牙胚分化的影响

目的：观察甲状腺旁腺激素（ｐａｒａｔｈｙｒｏｉｄ ｈｏｒｍｏｎｅ,ＰＴＨ）对体外培养１７ｄ的胎鼠下凳第一磨牙牙胚分化发育的影响,探讨ＰＴＨ在成牙本质细胞分化和牙本质形成中的作用。方法：器官培养、组织学观察和透射电本外培养的牙可出现顾牙本质细胞分化和牙本质形成。ＰＴ可促进成牙本质细胞的分化,能活跃状态。结论：牙胚培养在观察生长因子对牙齿发育的作用研究是有效的,ＰＴＨ在牙齿发育中具有一定的促进作用。     

巢蛋白在人牙齿发育过程中表达的免疫组化研究

目的:探讨巢蛋白(Nestin)在人牙齿发育过程中的表达模式和作用.方法:制备人牙齿发育各阶段以及人成牙本质细胞标本,采用SP法进行巢蛋白的免疫组织化学染色.结果:巢蛋白主要表达于功能性成牙本质细胞和成釉器.结论:巢蛋白参与人牙齿发育过程,特别是成牙本质细胞分化和基质分泌,可以作为功能性成牙本质细胞的标志物.     

牙本质磷蛋白在大鼠牙齿发育中表达的原位杂交研究

目的；观察牙本质磷蛋白(DPP)在大鼠牙齿发育各期的表达，探讨其在牙齿发育中的作用。方法：采用原位杂交方法，检测大鼠牙齿发育各阶段DPP mRNA的表达。结果：在分化成熟的大鼠成牙本质细胞中存在DPP mRNA表达，在前成釉细胞也存在DPP mRNA表达，在成釉细胞中可见较弱的表达，成牙骨质细胞和牙髓细胞呈阴性表达。结论：DPP的表达具有明显的时空特异性，它不但对牙本质的形成起重要作用，而且可能作为成釉细胞分化的信号分子调控牙釉质的形成。     

FAM20C在小鼠下颌髁突发育过程中的时空表达

目的 观察小鼠髁突发育过程的不同阶段,FAM20C在髁突软骨中的时空表达特点,试探究其在小鼠髁突发育过程中的可能作用机制。方法 苏木精-伊红(HE)染色和改良番红O-固绿染色观察胚胎17.5 d和出生后0、7、21 d 4组小鼠髁突软骨及软骨下骨的形态学变化。免疫组化染色观察相应时间点FAM20C在小鼠髁突软骨组织中的定位及表达。测量FAM20C阳性表达的平均光密度值,并进行单因素方差分析。结果 HE染色和改良番红O-固绿染色结果表明:随着软骨内骨化的进展,髁突软骨细胞层长度减少,软骨下骨体积增加,下颌髁突体积增大;免疫组化结果表明:4组小鼠髁突软骨组织中均有FAM20C阳性表达,FAM20C主要表达于髁突软骨增殖软骨细胞层和前肥大软骨细胞层,少量表达于肥大软骨细胞层及软骨下骨层,随着髁突发育,FAM20C表达逐渐减少。统计学结果显示,各时间点FAM20C阳性表达差异有统计学意义(P<0.01)。结论 FAM20C参与小鼠下颌髁突发育,可能通过调节髁突软骨细胞的增殖和分化在髁突形成中发挥重要作用。     

Inactivation of Fam20B in the dental epithelium of mice leads to supernumerary incisors

Tooth formation is tightly regulated by epithelial‐mesenchymal interactions via hierarchic cascades of signaling molecules. The glycosaminoglycan (GAG) chains covalently attached to the core protein of proteoglycans (PGs) provide docking sites for signaling molecules and their receptors during the morphogenesis of tissues and organs. Although PGs are believed to play important roles in tooth formation, little is known about their exact functions in this developmental process and the relevant molecular basis. Family with sequence similarity member 20‐B (FAM20B) is a newly identified kinase that phosphorylates the xylose in the common linkage region connecting the GAG with the protein core of PGs. The phosphorylation of xylose is essential for elongation of the common linkage region and the subsequent GAG assembly. In this study, we generated a Fam20B‐floxed allele in mice and found that inactivating Fam20B in the dental epithelium leads to supernumerary maxillary and mandibular incisors. This finding highlights the pivotal role of PGs in tooth morphogenesis and opens a new window for understanding the regulatory mechanism of PG‐mediated signaling cascades during tooth formation.     

Inactivation of Fam20b in the neural crest‐derived mesenchyme of mouse causes multiple craniofacial defects

The glycosaminoglycan (GAG) chains attached to the core proteins of proteoglycans exert multiple roles, such as enriching signal molecules and regulating the binding of ligands to the corresponding receptors. A newly identified kinase – family with sequence similarity 20 member B (FAM20B) – is essential for the formation of GAG chains. The FAM20B protein phosphorylates the initial xylose on the side chain of a serine residue in the protein. Although the GAG chains of proteoglycans are believed to be indispensable during craniofacial development, there are few reports on their exact functions in craniofacial organogenesis. In this study, by mating Wnt1‐cre mice with Fam20b‐floxed mice (Fam20bflox/flox), we created Wnt1‐Cre;Fam20bflox/flox mice in which Fam20b is ablated in the neural crest‐derived mesenchyme. The Wnt1‐Cre;Fam20bflox/flox mice died immediately after birth because of complete cleft palates. In addition to cleft palate, Wnt1‐Cre;Fam20bflox/flox mice also manifested tongue elevation, micrognathia, microcephaly, suture widening, and reduced mineralization in the calvaria, facial bones, and temporomandibular joint. These findings indicate that the proteoglycans formed through the catalysis of FAM20B are essential for the morphogenesis and mineralization of the craniofacial complex.     

Effects of Fam83h overexpression on enamel and dentine formation

Objective

The aim of this study was to determine if FAM83H over-expression causes dentine or enamel malformations.

Materials and methods

The full-length mouse Fam83h cDNA was inserted into the pCAGIG vector between a β-actin promoter and β-globin enhancer for ubiquitous expression in transgenic mice. Recombinant mouse FAM83H was expressed and used to generate polyclonal antibodies. Western blots showed enhanced expression of the Fam83h transgene. The effects of transgene expression on tooth development were assessed by microhardness measurements of enamel and dentine. Total thickness of incisor enamel at the level of the alveolar crest was measured and decussating rod patterns were visualized by scanning electron microscopy (SEM).

Results

Three transgenic mouse lines were selected based upon their transgene expression levels. There was no statistically significant difference in the Vickers microhardness values of enamel or dentine between the transgenic lines or between the transgenic lines and wild type mice. No statistically significant differences in enamel thickness were observed between the transgenic lines and the wild type mice. SEM analysis revealed no apparent differences in the enamel crystal and rod morphologies.

Conclusion

Our findings demonstrate that over-expression of FAM83H in mice does not produce a phenotype in dentine or enamel.     

Effects of maternal caffeine intake on the growth of rat tooth germs in protein-energy malnourished neonates

Fourteen rat dams with 8 pups each were fed either a 6, 12 or 20 per cent protein diet upon birth. Another group of 12 dams with the same number of pups was pair-fed either a 6, 12 or 20 per cent protein diet supplemented with caffeine (2 mg/100 g body weight). At day 15, randomly-selected pups were injected with [14C]-proline to determine collagen synthesis of the incisor and molar tooth germs. Another group of pups was used to determine calcium content of these tooth germs. Body weight, incisor weight and total calcium contents of tooth germs of pups from dams fed with 6 per cent protein diet were greater in the caffeine-supplemented group, whereas in the 20 per cent protein diet with caffeine group, these parameters were lower. The molar weights of the 12 per cent protein diet with caffeine animals were greater than the 12 per cent group without caffeine. The total hydroxyproline content of the incisor tooth germs from animals in the 12 per cent protein diet with caffeine was greater than is the non-caffeine group. However, total hydroxyproline of the molar tooth germs in the 20 per cent protein groups with caffeine was less than in the non-caffeine group. The rate of collagen synthesis of the incisor and molar tooth germs showed no difference in the presence or absence of caffeine in the 6, 12 and 20 per cent protein groups. Incisor and molar tooth germs are thus affected differently by the interaction of protein and caffeine, possibly due to differences in the pattern of tooth development.     

Developmental biology and genetics of dental malformations

The synthesis of tooth development biology with human studies focusing on inherited conditions that specifically interfere with tooth development is improving our understanding of normal and pathological tooth formation. The type of inherited dental malformations observed in a given kindred relate to when, during odontogenesis, the defective gene is critically expressed. Information about the protein encoded by the defective gene and the resulting dental phenotype helps us understand the major processes underway at different stages during tooth development. Genes affecting early tooth development (PAX9, MSX1, and AXIN2) are associated with familial tooth agenesis or oligodontia. Genes expressed by odontoblasts (COL1A1, COL1A2, and DSPP), and ameloblasts (AMELX, ENAM, MMP20, and KLK4) during the crown formation stage, are associated with dentinogenesis imperfecta, dentin dysplasia, and amelogenesis imperfecta. Late genes expressed during root formation (ALPL and DLX3) are associated with cementum agenesis (hypophosphatasia) and taurodontism. Understanding the relationships between normal tooth development and the dental pathologies associated with inherited diseases improves our ability to diagnose and treat patients suffering the manifestations of inherited dental disorders.