大鼠成釉细胞离体培养研究

目的：探讨成釉细胞离体培养方法及其特性。方法：采用贴壁培养法培养大鼠成釉细胞，用倒置显微镜观察细胞形态，用免疫组化检测细胞合成釉原蛋白的情况，用电镜观察成釉细胞离体培养时的形态。结果：大鼠成釉上皮细胞经消化酶解后原代培养于明胶包被的培养皿上，细胞贴壁良好，仅有少量成纤维细胞。在培养细胞生长一定时间后，主要为上皮型细胞成片生长，在细胞传至Ｆ２代时仍能分泌釉原蛋白。传至Ｆ５代时，细胞逐渐转变为长梭形，类似成纤维细胞。扫描电镜可见细胞突起，并有基质分泌。结论：用明胶作基质，对大鼠成釉细胞作离体培养，可以较长时间地维持其细胞特性。     

Differential Expression of the Tight Junction Proteins,Claudin‐1, Claudin‐4, Occludin,ZO‐1, and PAR3, in the Ameloblasts of Rat Upper Incisors

Tight junctions (TJs) create a paracellular permeability barrier to restrict the passage of ions, small solutes, and water. Ameloblasts are enamel‐forming cells that sequentially differentiate into preameloblasts, secretory, transition, and ruffle‐ended and smooth‐ended maturation ameloblasts (RAs and SAs). TJs are located at the proximal and distal ends of ameloblasts. TJs at the distal ends of secretory ameloblasts and RAs are well‐developed zonula occludens, but other TJs are moderately developed but incomplete zonula occludens (ZO) or less‐developed macula occludens. We herein examined the immunofluorescence localization of TJ proteins, 10 claudin isoforms, occludin, ZO‐1, and PAR3, a cell polarity‐related protein, in ameloblasts of rat upper incisors. ZO‐1 and claudin‐1 were detected at both ends of all ameloblasts except for the distal ends of SAs. Claudin‐4 and occludin were detected at both ends of transition and maturation ameloblasts except for the distal ends of SAs. PAR3 was detected at the proximal TJs of all ameloblasts and faintly at the distal TJs of early RAs. These results indicate that functional zonula occludens formed at the distal ends of the secretory ameloblasts and RAs consisted of different TJ proteins. Therefore, the distal TJs of secretory ameloblasts and RAs may differentially regulate the paracellular permeability to create a microenvironment suitable for enamel deposition and enamel maturation, respectively. In addition, PAR3 may be principally involved in the formation and maintenance of the proximal, but not distal, TJs. Anat Rec, 291:577–585, 2008. © 2008 Wiley‐Liss, Inc.     

Tumor suppressor in lung cancer-1 as a novel ameloblast adhesion molecule and its downregulation in ameloblastoma

Tumor suppressor in lung cancer-1 (TSLC1) is an intercellular adhesion molecule of the immunoglobulin superfamily. There is little information regarding the developmental expression profiles. In an attempt to clarify the distribution of TSLC1 proteins in mouse embryos tissue by immunohistochemistry, it was found that the TSLC1-specific signals were detected in the tooth germ as early as bud stage. The signals of TSLC1 were in the enamel epithelium at the cap stage, and became restricted to ameloblasts during the transition to and throughout the bell stage. In contrast, the signals for E-cadherin, which is important in odontogenesis, were distributed in all the components of the ectoderm-derived germ at any stage. In addition, E-cadherin preferred to locate on the basal membrane of ameloblasts, whereas TSLC1 preferred the lateral. And in further contrast, all the ameloblastomas examined were positive for E-cadherin (18/18) whereas all but one was negative for TSLC1 (1/18). These results indicate that TSLC1 is a novel interameloblast adhesion molecule that may be downregulated during ameloblastic tumorigenesis.     

不同时期成釉细胞氟损伤程度差异的动物实验研究

目的：GRP78和BCL-2在氟中毒大鼠不同时期成釉细胞中的表达和分布,探索氟牙症发病机制。方法：选择20只Wistra大鼠,饲喂氟浓度为75mg F-/L的自来水,8周后处死,通过HE染色和免疫组化技术观察大鼠成釉细胞形态和GRP78和BCL-2在氟中毒大鼠不同时期成釉细胞中的表达。结果：GRP78在成釉细胞分泌前期和分泌期表达高于转换期和成熟期,BCL-2在分泌期和转换期表达最高,在分泌前期和成熟期表达下降,各组间具有显著统计学差异（P<0.05）.结论：分泌早期和分泌期成釉细胞对氟诱导的内质网更加敏感,而在分泌期和转换期其抗凋亡能力更强。     

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。     

细胞极性相关蛋白CDC42和PAR3在小鼠牙胚发育过程中的表达

目的：研究细胞极性相关蛋白CDC42和PAR3在小鼠牙胚发育过程中的表达,探讨其在牙胚发育中的可能作用。方法：取13.5、14.5、16.5和18.5d（E13.5、E14.5、E16.5和E18.5）的小鼠胚胎以及出生后1和5d（PN1和PN5）的小鼠,分离头部,固定、脱钙、脱水、石蜡包埋、切片,HE染色观察牙胚的组织形态表现,采用免疫组织化学染色方法观察CDC42及PAR3在牙胚发育过程中的表达。结果：HE染色观察,E13.5~E18.5分别为小鼠牙胚发育的蕾状期、帽状期、钟状早期和钟状晚期,PN1小鼠的牙胚中可见分化成熟的成牙本质细胞和成釉细胞,PN5小鼠牙胚可见牙冠部发育完成。免疫组织化学染色,CDC42在E13.5、E14.5和E16.5小鼠牙胚中有广泛表达,在E18.5小鼠牙胚中表达较E13.5、E14.5和E16.5减少,在PN1和PN5小鼠牙胚中主要表达于成牙本质细胞和成釉细胞的分泌端;PAR3弱表达于E13.5和E14.5小鼠牙胚,在E16.5和E18.5小鼠牙胚上皮处表达明显增强,在PN1和PN5小鼠牙胚中表达较E18.5减弱。结论：CDC42和PAR3参与小鼠牙发育的过程,在牙胚发育早期可能参与小鼠牙胚的增殖及迁移,在牙胚发育晚期可能参与了成牙本质细胞和成釉细胞的分化,尤其在成牙本质细胞和成釉细胞极性的形成与维持中可能具有重要作用。     

Characteristics of the transverse 2D uniserial arrangement of rows of decussating enamel rods in the inner enamel layer of mouse mandibular incisors

The 2D arrangement of rows of enamel rods with alternating (decussating) tilt angles across the thickness of the inner layer in rat and mouse incisor enamel is well known and assumed to occur in a uniform and repetitive pattern. Some irregularities in the arrangement of rows have been reported, but no detailed investigation of row structure across the entire inner enamel layer currently exists. This investigation was undertaken to determine if the global row pattern in mouse mandibular incisor enamel is predominately regular in nature with only occasional anomalies or if rows of enamel rods have more spatial complexity than previously suspected. The data from this investigation indicate that rows of enamel rods are highly variable in length and have complex transverse arrangements across the width and thickness of the inner enamel layer. The majority of rows are short or medium in length, with 87% having < 100 rods per row. The remaining 13% are long rows (with 100–233 rods per row) that contain 46% of all enamel rods seen in transverse sections. Variable numbers of rows were associated with the lateral, central and mesial regions of the enamel layer. Each region contained different ratios of short, medium and long rows. A variety of relationships was found along the transverse length of rows in each region, including uniform associations of alternating rod tilts between neighboring rows, and instances where two rows having the same rod tilt were paired for variable distances then moved apart to accommodate rows of opposite tilt. Sometimes a row appeared to branch into two rows with the same tilt, or conversely where two rows merged into one row depending upon the mesial‐to‐lateral direction in which the row was viewed. Some rows showed both pairing and branching/merging along their length. These tended to be among the longest rows identified, and they often crossed the central region with extensions into the lateral and mesial regions. The most frequent row arrangement was a row of petite length nestled at the side of another row having the same rod tilt (30% of all rows). These were termed ‘focal stacks’ and may relate to the evolution of uniserial rat and mouse incisor enamel from a multilayered ancestor. The mesial and lateral endpoints of rows also showed complex arrangements with the dentinoenamel junction (DEJ), the inner enamel layer itself, and the boundary area to the outer enamel layer. It was concluded that the diversity in row lengths and various spatial arrangements both within and between rows across the transverse plane provides a method to interlock the enamel layer across each region and keep the enamel layer compact relative to the curving DEJ surface. The uniserial pattern for rows in mouse mandibular incisors is not uniform, but diverse and very complex.     

A developmental comparison of matrix metalloproteinase-20 and amelogenin null mouse enamel

Mutations in both the human amelogenin and human matrix metalloproteinase-20 ( MMP20 , enamelysin) genes cause amelogenesis imperfecta . Both genes have also been individually deleted from the mouse and each deletion results in defective dental enamel. Here, we compare the stage-specific progression of enamel development in continuously erupting mouse incisors from amelogenin null and MMP-20 null mice. Our goal was to closely examine differences in enamel and enamel organ structure between these mice that would allow a better understanding of each protein's function. The predominant feature of the amelogenin null incisors was the late onset of mineral deposition, with little or no protein present within the forming mineral. Conversely, the developing MMP-20 null incisors had a layer of protein between the apical surface of the ameloblasts and the forming enamel. Furthermore, the protein present within the enamel matrix was disorganized. An analysis of crystal structure demonstrated that the thin amelogenin null enamel was plate-like, while the MMP-20 null enamel had a disrupted prism pattern. These results suggest that amelogenin is essential for appositional crystal growth during the early to mid-secretory stage and for the maintenance of the crystal ribbon structure. They also suggest that MMP-20 is responsible for enamel matrix organization and for subsequent efficient reabsorption of enamel matrix proteins. Both genes are essential for the generation of full-thickness enamel containing the characteristic decussating prism pattern.     

Identification of secreted and membrane proteins in the rat incisor enamel organ using a signal-trap screening approach

The secretome represents the subset of proteins that are targeted by signal peptides to the endoplasmic reticulum. Among those, secreted proteins play a pivotal role because they regulate determinant cell activities such as differentiation and intercellular communication. In calcified tissues, they also represent key players in extracellular mineralization . This study was carried out to establish a secretome profile of rat enamel organ (EO) cells. A functional genomic technology, based on the signal trap methodology, was applied, starting with a library of 5'-enriched cDNA fragments prepared from rat incisor EOs. A total of 2,592 clones were analyzed by means of macroarray hybridizations and DNA sequencing. Ninety-four unique clones encoding a signal peptide were retrieved. Among those were 84 matched known genes, many not previously reported to be expressed by the EO. Most importantly, 10 clones were classified as being novel, with EO-009 identified as the rat homolog of human APin protein. These data indicate that many secreted and membrane-embedded EO proteins still remain to be identified, some of which may play crucial roles in regulating processes that create an optimal environment for the formation and organization of apatite crystals into a complex three-dimensional calcified matrix.     

细胞感受器初级纤毛在小鼠牙发育中的分布特征

目的:观察并总结初级纤毛在牙发育中的分布特征,揭示初级纤毛与牙发育的相关性.方法:进行小鼠牙胚组织切片苏木素-伊红(HE)染色和免疫荧光染色,观察初级纤毛在小鼠牙胚发育的蕾状期、帽状期、钟状期以及出生后细胞外基质分泌时期的分布范围和特点.结果:初级纤毛在蕾状期已广泛分布于牙胚上皮和间充质组织,但在上皮基底膜层几乎没有初...