环境毒物暴露大鼠下颌切牙釉质器官的显微解剖
1Institut National de la Santé et Recherche Médicale (INSERM) UMRS 1138, Paris-Diderot University, Pierre & Marie Curie University, Paris-Descartes University, Laboratory of Molecular Oral Pathophysiology,Cordeliers Research Centre, 2Unit of Formation and Research (UFR) of Odontology,Paris-Diderot University
Summary
了解搪瓷的形成和可能的改变需要研究成釉细胞活动。在这里, 我们描述了一个可靠和一致的方法, 显微解剖搪瓷器官含有分泌物和成熟阶段成釉细胞, 可用于进一步定量和定性实验程序。
Abstract
由于环境条件和生活方式造成的搪瓷缺陷是公共卫生关注的问题, 因为它们的发病率很高。这些缺陷是由负责搪瓷合成的细胞的活性改变所造成的, 成釉细胞是在搪瓷器官中存在的。在 amelogenesis 期间, 成釉细胞遵循一个特定的和精确的序列的事件的扩散, 分化和死亡。大鼠不断生长的门牙是研究成釉细胞活性和分化阶段的一种适宜的实验模型。在这里, 我们描述了一个可靠和一致的方法, 对暴露于环境毒物的大鼠的牙釉质器官进行显微解剖。微解剖的牙科上皮含有分泌物和成熟阶段的成釉细胞, 可用于定性实验, 如免疫组化分析和原位杂交, 以及定量分析, 如qPCR, RNA 序列, 和西方印迹。
Introduction
许多发展中的搪瓷缺陷可能是由于暴露于环境毒物和/或不适当的生活方式1,2,3,4。使用目前所描述的程序来破坏 amelogenesis 事件和分子的特性, 将促进使用由此产生的搪瓷缺陷, 作为暴露于几种毒物的早期标记, 并有助于重建健康史。每个病人在围产期期间, 搪瓷是综合升华 1, 2.搪瓷合成可分为四个主要阶段, 取决于成釉细胞活动5。第一步重整旗鼓前体细胞和成釉细胞的增殖。在第二步, 分化成釉细胞分泌搪瓷基质蛋白 (EMPs), 主要釉原蛋白, 釉蛋白和 ameloblastin, 这决定了最终搪瓷的厚度。因此, 电磁脉冲合成的任何干扰都会导致搪瓷的数量缺陷。在充分的搪瓷厚度的沉积以后, 成熟阶段开始。在这个阶段, 磷灰石微晶生长的宽度和厚度, 使搪瓷达到最高的矿化比发现在一个生物组织, 多达96% 的重量。破坏在成熟阶段发生的事件导致质量搪瓷缺陷。最后, 成釉细胞进入成熟后的阶段, 也称为啮齿动物的色素沉着, 在牙齿喷发过程中发生凋亡, 使搪瓷缺损 (如有) 无法挽回和不可逆转, 因此缺陷提供了潜在的回顾性记录。成釉细胞强调。在啮齿类动物中, amelogenesis 遵循类似的事件序列, 其门牙的特殊性不断增加, 这使得它们成为研究 amelogenesis 一般过程的合适模型。因此, amelogenesis 的任何中断都会导致搪瓷质量和/或数量的改变, 这取决于中断事件的时间窗口。在这个意义上, 暴露于二恶英, 铅和内分泌干扰化学品 (EDCs), 如双酚 a (BPA), 染料木黄酮和 vinclozolin, 已被证明生成搪瓷 hypomineralizations1,2,3 ,6,7,8。在胎儿期和出生后的第一个月内, 在低剂量 BPA 剂量的大鼠的门牙上发现不对称的白色不透明斑点1。这些搪瓷缺陷的大鼠, 以及人磨牙切牙 hypomineralization (MIH), 具有相似的临床, 结构, 和生物化学的特点。MIH 是最近被描述的牙釉质病理学, 病因仍然是不明的9,10尽管许多原因因素被假设了9,10,11 ,12。
另一重要的搪瓷 hypomineralization 病理由于环境因素是牙氟中毒 (DF), 这是由于过量氟化物吸收 (> 0.1 毫克/千克/天)13,14。氟化物的主要来源是饮用的水, 补充或自然富含氟化物。氟化物也经常被规定防止龋齿, 但预防剂量仅比毒性的50% 低 (≤0.05 毫克/公斤/天)。MIH 和 DF, 由于暴露于环境因素而引起的两个常见的病症, 可能呈现出共同的特征, 因为氟化物与其他有毒物质 (如 EDCs2 ) 的 hypomineralizing 效应增强, 因此需要加以表征。或阿莫西林15。
在不同分化阶段对成釉细胞的大鼠釉质器官进行显微解剖, 有助于了解分子的作用机制, 能够扰乱成釉细胞活性, 导致牙釉质缺损在牙齿喷发后确诊。换言之, 由于环境毒物的影响, 搪瓷基因表达的变化和搪瓷基质成分的改变, 使暴露于毒物的历史得以重建, 并促进对公众的环境安全监测。健康.
Protocol
在本研究中使用的所有动物都是按照法国农业部 (A-75-06-12) 的护理和使用实验动物的指导方针维持的。 1. 动物接触毒物 在执行本议定书之前, 获得必要的机构批准, 并确保遵守所有动物护理指南。 应用研究协议的设计, 允许不同实验组的组成, 以测试分子对成釉细胞分泌期和成釉细胞成熟期的影响。在这里, 四组雄性大鼠的组成取决于他们接触氟化物 (NaF) 组合或…
Representative Results
许多搪瓷缺陷, 如牙科氟中毒12,13, 可能是由于环境条件由于过量氟化物吸收或搪瓷 hypomineralization 类似 MIH 由于暴露在一些 EDCs1, 7,22。这些发育的搪瓷缺陷可在大鼠身上进行实验复制 (图 1)1,2<s…
Discussion
改变成釉细胞活动和/或扰乱成釉细胞的扩散, 分化和成熟过程导致不可逆的搪瓷缺陷, 反过来, 搪瓷缺陷的表征可能有助于发展对改变的理解成釉细胞活动在 amelogenesis 期间。因此, 对分离的搪瓷器官的研究是决定其起源、环境或遗传的病理事件的决定性因素。
此技术最初是由希勒et描述的。17和罗宾逊et 。18, 用于显示对搪瓷器?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作由巴黎狄德罗大学、法国国立卫生和医学研究研究所 (INSERM) 和法国牙科医生研究所 (IFRO) 资助。
Materials
| Bisphenol A | Sigma Aldrich, Saint Louis MO | 239658 | |
| formalin 10% | Sigma-Aldrich, Saint Louis, MO | HT5012 | |
| Tri-Reagent | Euromedex, France | TR118 | |
| RLT buffer | Qiagen, Les Ulis, France | 74126 | RNeasy Protect Mini Kit |
| Androgen receptor antibody | Santa Cruz Biotechnology, Santa Cruz, CA) | sc-816 | rabbit polyclonal antibody |
| PBS 10x | EUOMEDEX | ET330.A | |
| Sodium fluoride (NaF) | Sigma-Aldrich, Saint Louis, MO | S-1504 | |
| paraplast regular | Leica microsystems, Nanterre cedex, France | 39601006 | called was/parafin in the text |
| tissue OCT | VWR, Fontenay-sous-Bois, France | 411243 | |
| Extra Fine Bonn Scissors – Straight/8.5 cm | PHYMEP , Paris, France | 14084-08 | |
| Handle for Scalpel Blades – 12.5 cm | PHYMEP, Paris, France | 10035-12 | |
| Curved Scalpel Blade | PHYMEP , Paris, France | 10035-20 | |
| Dissecting Knife – Fine/Straight Tip | PHYMEP , Paris, France | 10055-12 | |
| Circle Knife | PHYMEP, Paris, France | 10059-15 | |
| scalpel blades n°11 Swann-Morton | VWR, Fontenay-sous-Bois, France | 233-0024 | |
| binocular lens | Leica biosystems, Nanterre cedex, France | MZFLIII |
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Citazione di questo articolo
Houari, S., Babajko, S., Loiodice, S., Berdal, A., Jedeon, K. Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants. J. Vis. Exp. (133), e57081, doi:10.3791/57081 (2018).