Calbindin D28k‐like immunoreactivity during the formation of the enamel‐free area in the rat molar teeth

Previous studies have demonstrated the presence of calbindin D28k in the ameloblasts derived from the inner enamel epithelium. The occlusal surfaces of the rodent molars partly lack the enamel covering, which is referred to as enamel‐free area (EFA). In the present study, we compared the immunohistochemical localization of calbindin D28k‐like immunoreactivity (CB‐LI) in the cells at the EFA (EFA cells) and ameloblasts of the rat molar teeth at the light microscopic level. CB‐LI was strong in the ameloblasts of the presecretory through the protective stages, while it was faint at the late secretory to transitional stages. However, some mature ameloblasts lacked the immunoreactivity. On the other hand, the majority of EFA cells showed distinct polarization and elongation that were absent in few cells at the early stage of EFA formation. At all stages, the EFA cells adjacent to the ameloblasts showed CB‐LI, however, some cells adjacent to the mature ameloblasts lacked the reaction. Intensive CB‐LI was demonstrated in EFA cells at the reduced enamel epithelium. These immunohistochemical findings suggest EFA cells have cytochemical properties similar to those of ameloblasts. Anat Rec 258:384–390, 2000. © 2000 Wiley‐Liss, Inc.     

细胞极性在牙发育中的研究进展

细胞极性是指由于极性蛋白复合物之间的相互协作或排斥，使细胞骨架、细胞器以及生物大分子等呈极性分布的特性。细胞各个部分实现不同生理功能需要细胞极性蛋白及分子的精密调控。细胞极性在牙发育中发挥着重要作用，特别对于成釉细胞和成牙本质细胞的分化和功能意义重大。文章就细胞极性及相关分子和通路在牙发育中的研究进展进行综述。     

���Դ�����������ϸ����̬Ӱ�켰�ʺ����׿��������о�

目的探讨氟斑牙的发病机制及褪黑素是否对氟斑牙的发生有拮抗作用。方法 2009年3—10月于中国医科大学公共卫生学院将40只Wistar大鼠随机分为6组,包括阴性对照组、低氟组、高氟组、低氟+褪黑素组、高氟+褪黑素组。建立氟斑牙动物模型,制作切片后行HE染色,光镜下观察各组大鼠切牙成釉细胞形态。结果单纯给氟组大鼠。出现牙面粗糙、棕白色相间横纹、白垩色改变,高氟组大鼠的氟斑牙改变较低氟组的改变典型;成釉细胞扭曲变形,正常的高柱状形态丧失,胞内出现空泡等。注射褪黑素组与单纯给氟组的切牙一般状态及成釉细胞形态、排列未见明显差异。结论氟对大鼠切牙的成釉细胞有毒性效应,饮水氟含量高所致氟斑牙症状加重。褪黑素对氟斑牙的发生是否有拮抗作用有待进一步研究。     

TGF‐beta1 and TGFBR1 are Expressed in Ameloblasts and Promote MMP20 Expression

Transforming growth factor‐beta (TGF‐beta) signaling exerts a wide spectrum of biological functions. To investigate TGF‐beta signaling in amelogenesis, we initially assessed the expression of TGF‐beta1 and TGF‐beta receptor 1 (TGFBR1) in developing teeth by immunohistochemistry. Both TGF‐beta1 and TGFBR1 were strongly expressed in secreting ameloblasts. Next, we studied the effects of TGF‐beta signaling on the expression of MMP20 and KLK4 mRNA using ameloblast‐lineage cells (ALC) in vitro. Our RT‐PCR study showed that TGF‐beta1, TGFBR1, and enamel matrix proteases (MMP20 and KLK4) were expressed in ALC. Following TGF‐beta1 treatment, the expression of MMP20 mRNA, but not KLK4 mRNA, was significantly upregulated. To further confirm the TGF‐beta signaling involvement in the MMP20 expression, we constructed the activated TGFBR1 vector and transfected the construct into ALC. The activated TGFBR1 notably promoted MMP20 expression, but had no obvious effects on the KLK4 mRNA expression. Our studies strongly suggest that TGF‐beta signaling involved in amelogenesis is partially mediated by regulating the expression of MMP20 mRNA. Anat Rec, 292:885–890, 2009. © 2009 Wiley‐Liss, Inc.     

成釉细胞微环境对胚胎干细胞特性的影响

目的研究胚胎干细胞诱导后成牙分化的可行性。方法使用成釉细胞无血清条件培养液诱导胚胎干细胞分化,观察诱导后细胞形态学变化以及体外检测基因表达和免疫表型分析。结果成釉细胞无血清条件培养液诱导胚胎干细胞后,拟胚体的外周有鹅卵石样的细胞,类似于上皮样的细胞生成。同时,诱导组细胞中可检测CK14、AMBN及AMGN mRNA的表达,DMP1和DSPP表达阴性,进一步免疫表型分析显示,诱导组胚胎干细胞表达CK14、AMBN及AMGN阳性。结论利用成釉细胞无血清条件培养液分泌的信号分子在体外模拟上皮的微环境,使得胚胎干细胞实现了向牙源性上皮细胞方向的表型转化。     

慢性氟中毒作用下小鼠切牙成釉细胞的改变

目的 通过观察慢性氟中毒作用下小鼠切牙成釉细胞形态学变化,探讨氟斑牙形成机制。方法培养制作小鼠氟斑牙动物模型,在不同时期肉眼观察各时期成活小鼠切牙颜色及釉质表面变化;45d后处死动物,HE染色,光镜观察小鼠切牙成釉细胞各个时期的细胞形态学变化。结果 随着小鼠日常饮用水中氟化钠浓度的增加和时间的延长,小鼠切牙氟斑牙形态学改变逐渐加重;小鼠切牙成釉细胞扭曲变形,正常的高柱状形态丧失,胞内空泡,大小不等的黑色颗粒,托姆斯突被异位物质压迫等细胞形态学改变逐渐加重。结论 氟对小鼠切牙的毒性效应在其发育初始阶段即已产生影响,并与时间和剂量有密切关系,从小鼠切牙发育初始阶段研究氟斑牙的致病机制可能会有所启示。     

Gene‐expression profile and localization of Na+/K+‐ATPase in rat enamel organ cells

The sodium pump Na⁺/K⁺‐ATPase, expressed in virtually all cells of higher organisms, is involved in establishing a resting membrane potential and in creating a sodium gradient to facilitate a number of membrane‐associated transport activities. Na⁺/K⁺‐ATPase is an oligomer of α, β, and γ subunits. Four unique genes encode each of the α and β subunits. In dental enamel cells, the spatiotemporal expression of Na⁺/K⁺‐ATPase is poorly characterized. Using the rat incisor as a model, this study provides a comprehensive expression profile of all four α and all four β Na⁺/K⁺‐ATPase subunits throughout all stages of amelogenesis. Real‐time PCR, western blot analysis, and immunolocalization revealed that α1, β1, and β3 are expressed in the enamel organ and that all three are most highly expressed during late‐maturation‐stage amelogenesis. Expression of β3 was significantly higher than expression of β1, suggesting that the dominant Na⁺/K⁺‐ATPase consists of an α1β3 dimer. Localization of α1, β1, and β3 subunits in ameloblasts was primarily to the cytoplasm and occasionally along the basolateral membranes. Weaker expression was also noted in papillary layer cells during early maturation. Our data support that Na⁺/K⁺‐ATPase is functional in maturation‐stage ameloblasts.     

成釉细胞的研究进展

成釉细胞是牙器官形成的关键细胞,国内外已成功离体培养出成釉细胞,成釉细胞合成和分泌的细胞外基质--釉原蛋白在釉质的形成中起着关键作用.成釉细胞的增殖和分化受到多种因素的调控.本文就成釉细胞的离体培养、釉原蛋白的研究及影响成釉细胞增殖和分化的因素作一综述.     

孕鼠急性氟中毒对胚胎牙胚发育的影响

【目的】 探讨孕鼠氟中毒对子鼠牙胚分泌期成釉细胞的影响。【方法】 给怀孕14d的孕鼠(C57BL／6N)昆明孕鼠腹腔注射10mg／kg氟化钠(浓度为5260μmol／L)，每日一次连续7d。到胚胎20d时，一部分孕鼠麻醉下剖腹取出胎鼠，并迅速分离下颌第一磨牙，行常规HE染色，观察牙胚成釉细胞的形态改变；另一部分孕鼠自然分娩，待子鼠下颌第一磨牙萌出后行硬组织切片观察，观察牙齿表面釉质的改变。【结果】 孕期氟中毒导致了胎鼠牙胚牙尖部分泌期成釉细胞下囊腔的形成；萌出后的牙齿在与囊腔相对应的牙尖部位出现釉质发育不全性缺损。【结论】 孕鼠氟中毒诱导了胎鼠分泌期成釉细胞下囊腔的形成，导致胎鼠氟斑牙的形成。     

Vitamin D and tissue non-specific alkaline phosphatase in dental cells

Dental epithelium comprises different cell populations, including ameloblasts and stratum intermedium cells. Ameloblasts are vitamin D targets, and at least five proteins undergo specific modulation of their expression following the addition of 1 α ,25(OH)₂ vitamin D₃[1 α ,25(OH)₂D₃]. Stratum intermedium cells have not been studied in any great detail regarding vitamin D impact. Interestingly, in these cells, the tissue non-specific alkaline phosphatase (TNAP) is overexpressed. On the other hand, TNAP is a reliable bone marker of vitamin D action, similar to calbindins in kidney and intestine, previously used for studies of vitamin D activity in ameloblasts. Here, TNAP expression and activity were investigated in vivo in the microdissected epithelium and mesenchyme of mandible incisors. Physiological doses of 1 α ,25(OH)₂D₃ injected in control rats failed to modify TNAP activity in both dental epithelium and mesenchyme. No significant differences were observed in the steady-state levels of TNAP mRNAs of dental tissues from wild-type and vitamin D nuclear receptor (VDRnuc)-deficient mice of the same litters. These data suggest that, in contrast to ameloblasts, stratum intermedium cells are not sensitive to 1 α ,25(OH)₂D₃. An explanation for such a responsiveness of stratum intermedium cells to 1α,25(OH)₂D₃ is proposed based on the respective expressions of both vitamin D receptors (VDRnuc and 1,25D₃-[MARRS]) and the Dlx2 homeobox gene.