Name: micro-dissection of enamel organ from mandibular incisor of rats exposed to environmental toxicants
Uploaded: 2018-03-29T13:30:00.000+00:00
Duration: 8 min 12 s
Description: Watch this Scientific Journal Video about Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants at JoVE.com

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。

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
<ol><li>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。</li>
	<li>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) 组合或不与 BPA (图 1A)2。所有动物在65天被观察和解剖 (图 1B)。</li>
</ol>2. 成年大鼠半下颌的解剖
<ol><li>弄死大鼠由 CO2窒息, 可选择地然后被斩首对分开的头从身体的其他。在专用框16中, 向2中的老鼠公开5分钟。</li>
	<li>使用 #11 手术刀去除下唇上的所有皮肤, 以便方便地接触下切牙。</li>
	<li>用同样的手术刀, 在两个较低的门牙之间进行切口轻微的压力, 下颌将被分成两半。</li>
	<li>用手术刀切开颞下颌关节, 将下颚分离, 用细镊子握住半下颌。</li>
	<li>用手术刀切开周围的软组织, 用刮刀除去所有的肌肉、肌腱和韧带, 直到骨头完全干净为止。</li>
</ol>3. 切牙的隔离17,18
<ol><li>将刀片平行于切牙的主要纵轴, 仔细地剃掉骨头。开始在骨脊附近的门牙尖端 (近端), 直到年底的门牙附近的颈部循环。切口运动从近端向远端方向进行。</li>
	<li>用手术刀将第一切口远端至颈环, 以除去半下颌的 gonion, 并允许在不损害宫颈环或成釉细胞分泌阶段的情况下轻易脱去门牙(图2中的红线)。</li>
	<li>在第二个磨牙下面做第二次切割, 并在切牙的骨和内侧表面插入手术刀。当所有的基底骨被解除, 旋转向外切牙, 并采取了小心, 以避免搪瓷器官组织损害使用细镊子。</li>
</ol>4. 双目镜下搪瓷器官的显微解剖
<ol><li>用吸管将200µL 盐磷酸盐缓冲液 (PBS 1X) 放在切牙上。用细镊子与唇面朝上。在组织的无色和橙色部分之间做手术刀标记。此标记对应于随后可观察到的底层白点 (图 2)。</li>
	<li>刮, 用挖掘机或等效工具, 细胞表面从手术刀标记到顶端末端对应于分泌阶段 (无色) 成釉细胞, 并且从手术刀标记到门牙尖端为成熟阶段 (橙色) 成釉细胞。</li>
	<li>移除与过渡阶段相对应的 2 mm 组织, 如前面描述的19所述。在搪瓷器官解剖时不要打开门牙, 以免被间质污染。</li>
	<li>将位于门牙顶端部分 (图 2) 的颈部环切开, 如前所述20所示。</li>
	<li>收集10% 福尔马林或溶解溶液进行进一步的调查 (请参阅材料表)。</li>
</ol>5. 收集分离的搪瓷器官组织进行进一步调查
<ol><li>将200µL PBS 1X 缓冲器放在切牙上。</li>
	<li>用 #11 手术刀刀片, 仔细地分离牙上皮细胞逐渐在缓冲, 分别为每个阶段在手术刀标记之前做的帮助。</li>
	<li>对于细胞 RNA 和蛋白质提取, 把组织的溶解溶液, 允许提取蛋白质和 rna (参见材料表)。 注: 通过转动试管中的组织挖掘机, 然后研磨细胞直至获得均匀的混合物, 制备高质量的 rna。该混合物已准备好 RNA 和蛋白质提取根据制造商的过程, 这是以前描述的2,8,17。通常, 每种制剂都能获得大约5-10 µg 总 rna 和150µg 总蛋白。</li>
	<li>对于组织学分析, 免疫组化 (IHC) 和原位杂交, 把细胞层在10% 福尔马林2小时, 然后存储在4摄氏度在 PBS 1X。组织可以嵌入蜡/石蜡或组织 OCT 的冰冻切片。</li>
	<li>对于 EMP 提取物, 用细镊子取门牙。在短时间暴露于空气 (轻微脱水) 后, 在切牙的唇表面的中间部分可以看到白色斑点, 这代表了搪瓷矿化的起始。这个白点对应于成釉细胞的过渡/早期成熟阶段, 所以使用它作为提取EMPs21 的指示器。</li>
</ol>

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作者没有披露的利益冲突。

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, 用于显示对搪瓷器官分化各个阶段的具体影响。然后, 它已适应 EMP 提取19,24,25。我们目前使用该程序分离的主要部分搪瓷器官和收集生物样品在不同的环境条件, 以分析 rna 和蛋白质 (可能内和超细胞蛋白) 使用定量和定性技术.
显微解剖的精确性要求在其应用前进行大量的训练, 以允许收集保存的搪瓷器官, 避免间质污染和任何组织破坏。而且, 老鼠和小鼠的成功是相当困难的。事实上, 最棘手的部分是保持组织形态学, 组织学和方向的 IHC 和, 而不是传统的技术, 通常在门牙上进行这两种方法。
与脱钙全半下颌相比, 使用显微解剖的搪瓷器官的主要优点之一是, 由于缺乏任何治疗后, 牙釉质 rna 和蛋白质的维护。显微解剖的搪瓷器官, 可直接用于所有分子生物学和生物化学技术, 而不选择或培养细胞。这个过程的好处是直接测量 RNA 和蛋白质水平, 反映他们的数量在特定成釉细胞在体内在生理或病理条件。另外, 一些毒物在一个分化阶段具体地行动, 与氟化物的情况一样, 在成熟阶段成釉细胞, 而不是在分泌阶段部分23, 24.对搪瓷器官的不同区域进行显微解剖, 可以研究某些毒物或特定基因的作用机制, 其作用可能在整个搪瓷器官或孤立细胞中无法检测到。事实上, 成釉细胞, 特别是有区别的, 很难收集和培养在体外, 这是证明了缺乏研究报告的主题。唯一可以被隔离的牙科上皮细胞是从颈环中成釉细胞和干细胞20。此外, 报告的造釉细胞细胞线, 主要是鼠HAT-729, 鼠标LS830, 刚果解放军31, 和人类AM-132, 往往失去了在体内的分化特征成釉细胞: 他们表示胞外基质蛋白 (EMPs) 如 ameloblastin 类似于分泌-阶段成釉细胞, 也 KLK4 或 SLC26A4/pendrin, 如成熟阶段成釉细胞 8, 但或多或少无法表达釉原蛋白和矿化基质生产搪瓷。口腔上皮干细胞和胚胎成釉细胞的分离可能是研究成釉细胞分化的第一阶段的一个选择, 但对终釉质成矿过程的研究不能以不同的方式进行。体内模型。amelogenesis 的最后一部分是搪瓷质量的决定因素。虽然牙齿发育的关键因素, 如 Bmp, Msx, 后代, 凹槽, 嘘, Wnt 是很好的描述33,34, 那些紧密参与搪瓷质量仍然是不太了解。搪瓷质量的关键因素的特征是破译龋齿和发现创新性的治疗或预防治疗龋齿, 这构成了一个主要的公共卫生问题, 作为 92% 20 到64岁的成年人在世界上的牙齿腐烂至少有一个龋齿35,36,37。
接触环境毒物的啮齿类动物能够破坏 amelogenesis 和改变搪瓷质量, 这可能是研究环境因素的良好模型。类似的程序也可用于直接或间接参与 amelogenesis 的相关基因的功能研究。显微解剖的搪瓷器官是一种合适的材料来表征毒物的作用机制和他们的目标基因在体内避免任何实验性的偏见。当发育性搪瓷缺陷的特征, 这种方法可以作为预测模型的新的污染物潜在的病理影响。

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.

<table><tbody><tr><td>Bisphenol A</td><td>Sigma Aldrich, Saint Louis MO</td><td>239658</td><td></td></tr><tr><td>formalin 10%</td><td>Sigma-Aldrich, Saint Louis, MO</td><td>HT5012</td><td></td></tr><tr><td>Tri-Reagent</td><td>Euromedex, France</td><td>TR118</td><td></td></tr><tr><td>RLT buffer</td><td>Qiagen, Les Ulis, France</td><td>74126</td><td>RNeasy Protect Mini Kit</td></tr><tr><td>Androgen receptor antibody</td><td>Santa Cruz Biotechnology, Santa Cruz, CA)</td><td>sc-816</td><td>rabbit polyclonal antibody</td></tr><tr><td>PBS 10x</td><td>EUOMEDEX</td><td>ET330.A</td><td></td></tr><tr><td>Sodium fluoride (NaF)</td><td>Sigma-Aldrich, Saint Louis, MO</td><td>S-1504</td><td></td></tr><tr><td>paraplast regular</td><td>Leica microsystems, Nanterre cedex,&amp;nbsp;France</td><td>39601006</td><td>called was/parafin in the text</td></tr><tr><td>tissue OCT</td><td>VWR, Fontenay-sous-Bois, France</td><td>411243</td><td></td></tr><tr><td>Extra Fine Bonn Scissors - Straight/8.5 cm</td><td>PHYMEP , Paris, France</td><td>14084-08</td><td></td></tr><tr><td>Handle for Scalpel Blades - 12.5 cm</td><td>PHYMEP, Paris, France</td><td>10035-12</td><td></td></tr><tr><td>Curved Scalpel Blade</td><td>PHYMEP , Paris, France</td><td>10035-20</td><td></td></tr><tr><td>Dissecting Knife - Fine/Straight Tip</td><td>PHYMEP , Paris, France</td><td>10055-12</td><td></td></tr><tr><td>Circle Knife</td><td>PHYMEP, Paris, France</td><td>10059-15</td><td></td></tr><tr><td>scalpel blades n&amp;deg;11 Swann-Morton</td><td>VWR, Fontenay-sous-Bois, France</td><td>233-0024</td><td></td></tr><tr><td>binocular lens</td><td>Leica biosystems, Nanterre cedex,&amp;nbsp;France</td><td>MZFLIII</td><td></td></tr></tbody></table>

micro-dissection of enamel organ from mandibular incisor of rats exposed to environmental toxicants

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。

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)1,2,7,23,24。大鼠半下颌骨包含一个由三臼齿分离的单切牙, 被称为空隙的间隙。不断生长的切牙包含两个矿化组织, 由成牙本质细胞产生的牙本质, 和成釉细胞所产生的珐琅与其他上皮细胞的搪瓷器官(图 2)。鼠门牙似乎是一个合适的模型来研究 amelogenesis, 与臼齿相反, 它包含所有的成釉细胞增殖/分化阶段贯穿整个动物的生命。
从大鼠切牙的显微解剖釉器官可以根据成釉细胞分化阶段, 在轻微脱水后出现的白点的帮助下分离, 这可能被用作分泌物之间过渡阶段的指示器--和成熟阶段19,21,25。显微解剖的搪瓷组织的质量可以检查三色马尾松染色 (图3A)。显微观察显示典型的搪瓷上皮细胞和成釉细胞栅栏。由于缺乏胶原蛋白绿色染色 (图 3A) 和表达式 (图 3B), 没有间充质污染。这些显微解剖组织可用于定性分析, 如 IHC (图 3C) 和, 以及定量分析, 如 RT-qPCR (图 3B), RNA 序列, 和西方印迹。例如, 雄激素受体是专门在成熟阶段成釉细胞使用针对这种蛋白的特定抗体 (见材料表)(图 3C)8。主要成釉细胞分化阶段以特异基因表达模式26 为特征, 可在显微解剖的搪瓷器官上鉴定。例如, 分泌阶段成釉细胞表达釉蛋白2和成熟阶段成釉细胞表达激肽酶 4(KLK4) (图 3B)。它们含有高水平的铁蛋白, 它赋予了储存大量铁的能力, 这对搪瓷27的橙色颜色负责。
amelogenesis 的氟化物中断可以很容易地跟随观察细胞裂解物的 RNA 提取: 控制成熟阶段裂解物是橙色的, 而氟化物处理的是淡黄色到无色(图 4A)。从显微解剖成熟阶段搪瓷器官中提取的 RNA 水平分析显示, KLK4 对氟化物处理 (图 4B) 进行了下规, 最近在2中报告的大规模 transcriptomic 分析也证明了这一点。由分泌物或成熟阶段成釉细胞表达的特定蛋白质也可以使用显微解剖的搪瓷器官28 进行局部化。例如, 雄激素受体是专门使用显微解剖搪瓷器官8,28。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57081/57081fig1.jpg"> 图 1: 在使用双酚 a (BPA) 的情况下, 对暴露于氟化物 (NaF) 的大鼠的搪瓷缺陷进行观察.(A) 对本研究构成的四实验组的示意图表示, 这些组依赖于适用于大鼠的不同环境条件。从妊娠日 1 (fecondation) 直到断奶日 21 (P21), 5 µg/千克 BPA 在0.5 毫升的玉米油由饲每日口服给孕妇和哺乳女性, 而仅用玉米油管理的控制组。断奶后, 每座大坝都被发现并随机分布在相应的两组中之一。为这项研究选择了雄性大鼠, 仅暴露于5µg/千克/日 BPA 单, 5 毫米 NaF 单独, 或两者在同一剂量, 直到65天出生后 (P65)。对照组单独给予溶剂。(B) 在产后 30 (P30), 长期接触低剂量 BPA 的大鼠表现为白色不透明斑点, 并发现成年大鼠 (P65) 的牙齿表型不再明显。用 NaF 治疗大鼠典型牙氟中毒 (DF) 的交替白色和橙色带。在两种药物接触的大鼠中, 发现了一种具有重要切牙变色特征的更严重的表型。均质橙切釉典型对照大鼠。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57081/57081fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57081/57081fig2.jpg"> 图 2: 过程的示意图表示形式。从显微解剖搪瓷器官中分离成釉细胞的分泌物和成熟阶段。大鼠半下颌骨含有三磨牙和持续生长的切牙, 两个矿化组织, 由成牙本质细胞 (黄色) 产生的牙本质, 以及成釉细胞 (橙色) 产生的搪瓷。当切牙被分离和轻微脱水, 在唇表面顶端部分附近的白色斑点是可观察的。它有助于识别成釉细胞阶段的分化, 因为它涵盖了过渡阶段之间的分泌-和成熟阶段。搪瓷器官仔细细解剖, 根据成釉细胞分化阶段分离3个部分, 并用于定性 (组织学) 分析或定量实验方法 (mRNA 和蛋白质分析的 RT-qPCR 和西方印迹, 分别)。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57081/57081fig3.jpg"> 图 3: 显微解剖分泌物的质量控制和搪瓷器官的成熟阶段.大鼠搪瓷器官分离3个部分, 包含颈环, 分泌阶段成釉细胞, 成熟期成釉细胞。(A) 三色马尾松显微解剖部位的染色显示出不同的上皮细胞和无间充质细胞。缩放条, 100 µm. (B) 从分泌物和成熟期搪瓷器官中提取 rna 的 RT qPCR 实验进行基因表达分析。釉蛋白和 KLK4 表达的典型模式, 以及缺乏胶原蛋白1预期在间充质细胞。(C) 荧光染色使用抗体 (见材料表), 专门针对雄激素受体, 仅由成熟阶段成釉细胞表达。缩放条, 20 µm.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57081/57081fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57081/57081fig4.jpg"> 图 4: 经显微解剖的大鼠搪瓷器官获得的代表性结果.(A) 当细胞被悬浮在裂解缓冲器中进行 RNA 提取 (见材料表) 时, 含有色素成釉细胞的成熟阶段组织在控制和 BPA 处理的条件下出现橙色。分泌阶段部分是黄色的不管情况。在 NaF 治疗中, 所有的裂解物都是淡黄色到无色。(B) 以各种环境条件提交的大鼠搪瓷器官的 RNA 制剂所获得的 RT qPCR 数据的示例。NaF 下调 KLK4 在成熟阶段, BPA 增加釉蛋白表达在分泌阶段。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57081/57081fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants at JoVE.com

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

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。

环境毒物暴露大鼠下颌切牙釉质器官的显微解剖

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

micro-dissection-enamel-organ-from-mandibular-incisor-rats-exposed-to

Nanyang Technological University

Research

JoVE Journal

Medicine

10.0K 次观看.  Cordeliers Research Centre. 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。

视频: Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

<meta charset="utf8"></head><body>评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

科研

环境科学

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

使用Trowell型器官培养研究牙科干细胞的调节

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

发育生物学

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

从小鼠和人类牙齿中快速分离单个细胞

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

将人类牙齿中的类器官确立为机械研究和再生疗法的有力工具

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

通过双光子显微镜实时跟踪牙齿细胞

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

作者聚焦:了解成年小鼠牙齿组织更新和修复中的动态细胞行为 

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

牙齿和呼吸细胞及器官在微重力下的繁殖

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

生物工程

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

环境毒物暴露大鼠下颌切牙釉质器官的显微解剖

摘要

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评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

使用Trowell型器官培养研究牙科干细胞的调节

从小鼠和人类牙齿中快速分离单个细胞

将人类牙齿中的类器官确立为机械研究和再生疗法的有力工具

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

牙齿和呼吸细胞及器官在微重力下的繁殖

从成年小鼠切牙牙源性上皮干细胞的分离和培养

环境毒物暴露大鼠下颌切牙釉质器官的显微解剖

摘要

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评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

使用Trowell型器官培养研究牙科干细胞的调节

从小鼠和人类牙齿中快速分离单个细胞

将人类牙齿中的类器官确立为机械研究和再生疗法的有力工具

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

牙齿和呼吸细胞及器官在微重力下的繁殖

从成年小鼠切牙牙源性上皮干细胞的分离和培养

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动