过量氟对鼠牙釉质发育的影响

为了探讨过量氟对鼠牙釉质发育的影响，本研究以大白鼠为实验动物，观察氟中毒后鼠牙造釉器，造釉细胞和釉质的组织形态学变化，并利用生化方法，检测氟中毒后釉质分泌期和成熟期釉质蛋白含量的变化。结果显示分泌期造釉细胞有空泡形成，釉基质钙化不均。分泌期釉质蛋白含量未见明显改变。成熟期釉质帽白含量增加。     

氟斑牙的发病机理

氟斑牙的损害表现为釉质表面过度钙化及表面下层钙化不全。氟对造釉细胞的各期均有实质性的损害改变；通过对釉质蛋白醇、造釉细胞的代谢、釉基质ＰＨ值，釉质晶体形成及以造釉细胞调节功能的影响，而使成熟期釉质中蛋白质潴留，阻碍羟基磷灰石晶体的形成。这些因素都都会使牙釉质受到损害。本文就氟对造牙本质细胞的影响作了简要介绍。     

氟斑牙的发病机理

氟斑牙的损害表现为釉质表面过度钙化及表面下层钙化不全。氟对造釉细胞的各期均有实质性的损害改变;通过对釉质蛋白酶、造釉细胞的代谢、釉基质pH值、釉质晶体形成以及对造釉细胞调节功能的影响,而使成熟期釉质中蛋白质潴留,阻碍羟基磷灰石晶体的形成,这些因素都会使牙釉质受到损害。本文就氟对造牙本质细胞的影响也作了简要介绍。     

孕鼠急性氟中毒对胎鼠牙胚造釉细胞影响的超微结构研究

目的 :建立孕鼠急性氟中毒动物模型 ,观察孕鼠急性氟中毒对胎鼠牙胚造釉细胞的影响。方法 :孕 14d的C5 7BL/6J鼠 ,实验组腹腔注射 10mg/kg·wt氟化钠 ,对照组注射等量生理盐水。每天注射一次 ,直至孕 2 0d。麻醉下处死孕鼠 ,取出胎鼠 ,分离下颌第一磨牙 ,进行光镜超微结构切片观察。结果 :孕鼠大剂量的氟可以引起胎鼠造釉细胞下囊腔的形成。囊腔上方的造釉细胞严重受损 :核上区线粒体明显肿胀或溶解 ,囊壁的造釉细胞细胞质和细胞核显示各种程度改变 ,细胞核染色质浓缩 ,凋亡小体出现 ,胞浆从细胞中脱出进入囊腔 ,囊腔中可见细胞及细胞碎片 ,异常分泌的釉基质 ,吞噬细胞 ,托马斯突变短或消失 ,囊腔下方的釉质分泌减少或终止。结论 :大剂量的氟不仅能引起发育中的鼠牙胚氟斑牙的形成 ,而且也能引起胎鼠牙胚氟斑牙的形成。氟引起胎鼠牙胚造釉细胞下囊腔的形成 ,囊腔上方的造釉细胞是以细胞凋亡的方式进行退变的。     

高氟环境对小鼠牙齿发育中CD44表达的影响

目的：探讨CD44在正常牙齿发育以及高氟环境中牙齿发育表达变化,从细胞粘附分子与发育学角度揭示氟牙症发病机制。方法：建立小鼠急性氟中毒动物模型,采用免疫组织化学的方法,观察CD44在正常及高氟环境中下颌第一磨牙牙胚发育的钟状形态发生期、分化期、分泌期、成熟期的表达。结果：钟状形态发生期、钟状分化、分泌期实验组和对照组成釉器中间层细胞CD44的表达水平无差异。成熟期实验组CD44的表达水平显著低于对照组。结论：过量氟影响成熟期中间层细胞CD44的表达,使其表达下调,推测影响釉质的矿化。     

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.     

高氟环境对小鼠牙齿发育中E-钙黏附素表达的影响

目的:探讨E-钙黏附素在正常牙齿发育以及高氟环境中牙齿发育的表达变化,从细胞黏附分子与发育学角度揭示氟牙症发病机制.方法: 建立小鼠急性氟中毒动物模型,采用免疫组织化学方法,观察E-钙黏附素在正常及高氟环境中下颌第一磨牙牙胚发育的钟状形态发生期、分化期、分泌期、成熟期的表达.结果:钟状形态发生期实验组和对照组成釉细胞E-钙黏附素的表达水平无差异;钟状分化、分泌期实验组E-钙黏附素的表达水平显著低于对照组;钟状成熟期实验组E-钙黏附素的表达水平低于对照组.结论:过量氟摄入后影响牙胚成釉细胞E-钙黏附素的表达,导致釉质发育紊乱,形成多孔的结构.这可能是氟牙症的发病机制之一.     

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。     

过量氟对大鼠下切牙发育过程中釉蛋白表达的影响

目的 研究过量氟对大鼠下切牙发育过程中釉蛋白表达的影响。方法 选择20只Wistar大鼠,随机分成实验组和对照组。饲养8周后处死,利用HE染色和免疫组织化学染色的方法观察过量氟对大鼠成釉细胞形态及釉蛋白表达的影响。结果 实验组大鼠下切牙成釉细胞由原有的高柱状结构变矮,细胞排列成多层,釉基质形成混乱,成釉细胞和成牙本质细胞中釉蛋白表达明显低于对照组,其差异有统计学意义（P<0.01）。结论 过量氟可抑制釉蛋白表达,影响釉质发育。     

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。     

过量氟对大鼠切牙成釉细胞增殖与凋亡的影响

目的　研究氟牙症的发生机制。方法　选择 2 0只Wistar大鼠 ,随机分为 2组 :Ⅰ组 (对照组 )和Ⅱ组 (5 0mg/LF-)。 8周后处死动物 ,利用磨片、HE染色、核仁形成区嗜银染色 (AgNORs)和原位细胞凋亡检测 (TUNEL)技术观察过量氟对大鼠切牙形态及机能的影响。结果　Ⅱ组大鼠切牙釉质生长线明显 ,分泌期成釉细胞嗜伊红颗粒蓄积 ,分泌前期成釉细胞AgNORs颗粒数低于Ⅰ组 ,差异有显著性 (P <0 0 0 1) ,成釉细胞凋亡增多 ,并伴有凋亡现象向分泌期迁移。结论　过量氟可抑制成釉细胞增生 ,促进成釉细胞凋亡 ,为氟牙症发生的一种机制     

Changes in the fluoride-induced modulation of maturation stage ameloblasts of rats

The maturation stage of enamel development is characterized by a cyclic modulation of the ameloblasts between bands of smooth-ended cells and longer bands of ruffle-ended cells. There are cyclic patterns of calcein staining of and 45Ca uptake in the enamel associated with this cellular modulation. Rats were given 0, 75, 100, or 150 ppm fluoride in their drinking water. Fluoride disrupted the cyclic patterns of the maturation stage, resulting in fewer bands of smooth-ended ameloblasts, fewer calcein-stained stripes, and fewer cycles of 45Ca uptake. When animals were given water containing 0 ppm fluoride following ingestion of water containing 100 ppm fluoride, the pattern of calcein staining returned to that of the control enamel. The disruption of the cyclic patterns in the maturation stage and the increased protein content of maturation enamel seem to be among the early events in the development of fluorosis.     

过量氟对大鼠切牙成釉细胞Cbfα1表达的影响

目的:了解过量氟对大鼠切牙核心结合因子(Cbfα1)蛋白表达的影响,从蛋白水平探讨氟斑牙的发病机制.方法:20只Wistar大鼠,随机分为实验组(饮用100mg/L氟化水)和对照组(饮用蒸馏水),饲养8周后,处死大鼠,获取切牙标本,通过免疫组化染色进行观察,比较Cbfα1在2组大鼠切牙成釉细胞中的表达情况,采用Axioplan 2imaging显微图像分析系统和SPSS10.0软件包分别进行图像和数据统计分析.结果:免疫组化结果显示,Cbfα1在分泌期的成釉细胞胞核中明显表达,实验组阳性率(35.28±1.20)%显著高于对照组(14.41±4.07)%,差异有显著性(P＜0.01).结论:过量氟有可能引起Cbfα1蛋白在分泌期成釉细胞中的表达增多,从而在某种程度上影响釉质的矿化和发育,导致釉质发育障碍.     

High amounts of fluoride induce apoptosis/cell death in matured ameloblast-like LS8 cells by downregulating Bcl-2

Objective

Excessive fluoride intake during enamel formation may result in enamel fluorosis and apoptosis is regarded to be involved in the process by an unclear mechanism. We hypothesize that excessive fluoride might cause apoptosis in the ameloblasts and fluoride-induced apoptosis varies with the maturation stages of ameloblasts.

Methods

We set up an in vitro differentiation model of ameloblasts by using retinoic acid (RA) and dexamethasone (DEX) to induce the maturation of mouse ameloblast-like LS8 cells.

Results

The mRNA and protein levels of two enamel matrix proteins and two enamel proteinases were downregulated and upregulated, respectively, in the RA/DEX induced cells, indicating RA/DEX induced cells possessed the characteristics of matured ameloblasts. The strengthened endocytosis function and decreased intracellular pH value inside RA/DEX treated ameloblasts confirmed the maturation inducing effect of RA/DEX on ameloblasts. Excessive fluoride inhibited cell proliferation of ameloblasts within 72 h. High amounts of fluoride also induced more apoptosis/dead cells and reduced the expression of Bcl-2, but to a different degree in the non-induced cells and RA/DEX induced cells.

Conclusions

We inferred that high doses of fluoride may easily target the transitional/early maturation ameloblasts and cause apoptosis or cell death. Bcl-2 might be involved in this process.     

Enamel mineralization in the absence of maturation stage ameloblasts

The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify to which extent enamel mineralizes in the absence of these cells. Maturation stage ameloblasts and adjacent dental follicle cells from rat lower incisors were surgically removed and the limits of this removal were marked by notches made in the enamel. Histological analysis confirmed that the ameloblasts had been removed within the limits of the notches. The teeth erupted and when the notches appeared in the mouth, the enamel in the experimental teeth was hard but whitish compared to the yellowish colour of the contralateral incisors used as control. SEM images revealed similar enamel rod arrangement in both groups. Decreased mineral content was observed in some specimens by polarized light microscopy, and microhardness values were much lower in the experimental teeth. FTIR analysis showed that higher amounts of protein were found in most experimental teeth, compared with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels, suggesting that most of them were below 5 kDa. These results suggest that the enamel matured in the absence of ameloblasts has increased protein content and a much lower mineral content, suggesting that maturation stage ameloblasts are essential for proper enamel mineralization.     

过量氟对大鼠切牙釉丛蛋白表达的影响

目的研究过量氟对大鼠切牙发育过程中釉丛蛋白表达的影响,探讨氟斑牙的发病机制。方法选择20只Wistar大鼠,随机分成两组:对照组(蒸馏水组)和实验组(100mg/LF-)。复制大鼠氟斑牙模型。饲养8周处死动物,利用免疫组化和实时荧光定量PCR(Real-TimePCR)方法观察过量氟对大鼠切牙中釉丛蛋白表达的影响。结果免疫组化结果显示釉丛蛋白在分泌前期和分泌期的成釉细胞中呈阳性表达。实验组釉丛蛋白的表达明显弱于对照组,存在显著性差异(P<0.01)。Real-TimePCR结果显示釉丛蛋白mRNA在对照组表达明显高于在实验组表达水平(P<0.01)。结论过量氟可能通过抑制釉丛蛋白的表达,从而影响釉质的矿化,导致釉质发育障碍。     

Composition of mineralizing incisor enamel in cystic fibrosis transmembrane conductance regulator‐deficient mice

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr‐null mice by quantitative electron probe microanalysis. Maturation‐stage enamel from Cftr‐null mice contained less chloride and calcium than did wild‐type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr‐null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr‐deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.     

Ultrastructural examination of various stages of amelogenesis in the rat following parenteral fluoride administration

The stages of differentiation, formation, transition, and early maturation were examined following multiple high dosages of sodium fluoride. The ability of the cells to recover from the effects of the fluoride ion was also studied. Fluorotic differentiating and transitional amelohlasts exhibited no detectable morphological differences from the control cells.The formative stage demonstrated a response to the fluoride in the morphology of the cells and their product. The ameloblast contained unusual vacuoles and granules and the adjacent forming enamel contained paired hypermineralized and hypomineralized zones. During maturation, groups of the ameloblasts often were altered with huge intracellular vacuoles, while adjacent enamel appeared to mature normally.Animals killed after a recovery period demonstrated no cellular disturbances and a layer of normal enamel formed over the fluoride response.