Name: zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing
Uploaded: 2015-02-25T17:00:00
Description: Watch this Scientific Journal Video about Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing at JoVE.com

这项工作是由卫生科学中西部大学基金研究促进格兰特授予英皇酒店与研究中西部大学办公室和赞助节目校内格兰特授予KJL支持。

该协议符合AVMA动物福利原则实验动物的照顾和使用，已被批准的机构动物护理和使用委员会（IACUC）在中西部的大学。 1.准备麻醉，设备，及媒体<ol><li>脱氯约1.5升使用一个商业脱氯剂（可在宠物商店），或者通过让水静置24小时自来水。获得3 1升的烧杯中，并填写每用500毫升水脱氯。标签之一任何操作之前，拿着鱼和一个用于恢复。到第三烧杯中，加入三卡因甲磺酸盐的10...

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对于角膜移植培养的成功的最关键的步骤是使刻度附着到培养皿约2 - 除了培养基的前3分钟。大约鳞的75％将附着和生长片材（将需要建立每个实验培养物的数量，以进行相应的调整）。角膜片不形成围绕每个尺度从鱼取出，并放置在培养有几个原因。首先，外植体的DAPI染色揭示了一些鳞有少数细胞附着，因此，预计这些尺度不会有角膜的表。其次，外植体要坚持组织培养皿的能力是板材迁移的?...

<html>细胞运动性的​​研究已经传统上集中于单独迁移细胞。角膜从鱼类和两栖类植培养已证明是一个功能强大的模型系统使用实验和数学建模方法1-6来研究细胞运动的机制。上单独迁移细胞的实验工作是由细胞的快速，平稳的滑行动作的帮助下，同时保持一个统一的形状，速度和方向。然而， 在体内 ，细胞频繁移动的统称，同时保持细胞-细胞连接，尤其是在胚胎发育，伤口愈合，和转移。因此，集体细胞迁移是研究细胞迁移的领域内不断增长和日益突出的区域。这些细胞的迁移集体最近被描述7，它有很多独特的功能，这使它成为一个强大的系统来研究集体细胞迁移的伤口愈合模型。 ove_content“&gt;当秤从麻醉的成年斑马鱼弹拨，一些上皮组织保持附着到刻度的底面。当细胞培养基中加入，这些上皮细胞，或角膜，快速地迁移至自规模集体单元的外植体培养物可被看作是一种上皮伤口愈合模型中，其中细胞远离外植体，因为它们会在整个伤口床，以重新建立完整的上皮的临时基质迁移。最近的数据表明，这种体外培养模仿的许多特征在体内在成年斑马鱼伤口愈合。伤口闭合率8转换为类似于在体外系统7观察到的迁移速度，另外， 在体内伤口愈合模型中，与华法林治疗表明，止血对率没有影响愈合，而用氢化可的松治疗，以降低免疫反应不耽误表皮细胞再生8 </SUP&gt;。作为当秤从动物取出的斑马鱼不流血，止血是不是一个主要参与者。虽然我们已经发现免疫细胞外植体，我们还没有研究它们可能对集体细胞迁移的影响。 这里介绍的协议描述了如何建立斑马鱼角膜外植体培养，以确保在许多生物测定使用的一致性和可重复性。这些外植体培养在集体细胞迁移的体外模型中特别引人注目的几个原因。首先，斑马鱼角膜是建立植文化小时内使用原代细胞。因此，这些细胞未经历与主细胞9-13的通道相关联的形态和基因表达的变化。其次，该系统是一款型号为表皮细胞再生的响应研究伤人14，作为回应伤人，上皮间质潮流的一部分启动ansition（EMT）的过程就证明了基因表达的变化和细胞骨架重排14,15。因此，发生在未处理的细胞中的基因表达和运动的背景变化已被鉴定并提供了一​​个上下文来解释与治疗的变化。另外，鱼角膜和人类角质形成细胞在功能上是等效的;两者均为各自物种的初级上皮细胞和播放在上皮伤口愈合的关键作用。第三，成年斑马鱼正获得承认作为模型系统，适用于各种包括黑素瘤和其它癌症19-21，皮肤伤口愈合8,14和组织再生22-24人类疾病16-18中。 这个模型系统的技术优势也同样引人注目。可从非营利性的，集中的设施越来越多的突变和转基因株系。具体而言，斑马鱼突变项目（ZMP）在威康信托基金会桑格研究所的目标是创建在每个蛋白质编码基因的斑马鱼基因敲除等位基因。目前，他们已经突变11892个基因，基因组的大约45％，具有24088的等位基因特征。另外，ZMP接受建议，并会产生剔除装置收费。最近的基因敲除的方法在幼虫和成年斑马鱼25-28提供了灵活性，研究发育的重要基因，这些基因转基因方法是有问题的或不切实际的功能。 由于它们的非常迅速的〜145微米/小时运动速率建立培养物29后不久，测定可以迅速地完成（一般在24小时以下）。由于实验可以迅速完成，文化发展以及在室温，用几个涉及视频显微镜哺乳动物细胞迁移试验是避免相关的技术障碍。此外，在紧缩研究预算的年龄，斑马鱼肥胖是容易且廉价地维护。 外植体的结构和行为是复杂的。作为当比例尺被去除鱼不流血，这是不可能的比浅表皮层得多的规模被除去。角膜细胞似乎是主要的细胞类型的外植体时秤最初从鱼除去细胞的绝大部分出现染色用抗E-钙粘蛋白抗体14。此外，没有任何证据表明在初始培养的成纤维细胞作为判断不存在波形蛋白染色通过免疫荧光14。但是，随着反复除垢，似乎有在植众多嗜中性粒细胞（见视频1）。当暴露于LPS我们已经确定了这些细胞作为基于它们的大小，迅速地运动，并且其迁移出电池板的形态观察结果的中性粒细胞（数据未示出）。此外，这些细胞染色鲜艳的智慧h的抗体，以嗜中性粒细胞胞质因子，以及与各种次级IgG抗体，这表明它们具有在其表面上丰富FcR的（数据未示出）。多个细胞层是外植体中存在。共焦显微镜揭示邻近前缘的单个层的存在，以在靠近刻度角膜的两个以上的层，嗜中性粒细胞可见上方，下方，和之间的细胞角膜片。 在早期时间点，将细胞在多层植两极分化的边缘，引发角膜的集体的迁移，从外植体以约145微米/小时的初始速率。涵盖的外植体的面积急剧增加，从而导致细胞的片内扩散，张力，并前进前缘的下降速率。领导者和从动单元之间快速互变是在前缘的自发形成的孔的片材7内的形成和封闭过程中观察到的。由于与外植体开始的EMT过程继续，片材片段和角膜细胞失去其专业化，快速运动。于7天培养的基因表达，并与EMT一致的形态变化，伤口愈合，和炎症反应发生植正在考虑可行大约10天14。

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			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
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			<td height="63" style="height:63px;width:267px;">35 mm Glass Bottom Culture Dishes (Poly-D Lysine Coated)&#160;</td>
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zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing

由于其独特的游动性，鱼角膜分离从外植体培养物早已被用于研究单个细胞迁移的机制。然而，当外植体建立，这些细胞还集体移动，保持了许多这使得个体角膜一个有吸引力的模型来研究移民的特点：蠕动较快速度，广泛的肌动蛋白丰富的片层具有垂直肌动线，相对恒定的速度和迁移的方向。在早期的外植体，细胞与那些迁移统称单独迁移的快速互允许的细胞 - 细胞粘连在确定迁移模式中的作用的研究，并强调迁移的两种模式之间的分子联系。在后来的外植体的细胞失去其迅速地和集体迁移能力，上皮至间质转变发生和伤口愈合和炎症相关的基因的差异表达。因此，角膜外植体可以作为一种体外模型为皮肤创伤愈合过程中发生，并且可以代表一个独特的系统来研究集体细胞迁移的机制中所定义的基因表达的变化方案的情况下的表皮细胞再生。多种突变体和转基因斑马鱼线是可用的，其允许外植体可以从鱼具有不同遗传背景的确立。这允许不同的蛋白质，这些过程中的作用，以唯一地寻址。这里所概述的协议描述一种简单而有效的方法，用于在各种相关的集体细胞迁移测定的建立这些外植体培养中使用。

如示于图1中 ，角膜的片可以观察到从规模下建立的外植体培养的小时内迁移出去。有时，单独移植角膜已经从集中迁移片剖开可以观察到。另外，作为外植体是从已被预先弹拨鱼成立，有丰富的嗜中性粒细胞通过片材迁移。在较长的文化时期，角膜板碎片为细胞进行EMT 14。 片前进的初始速率是极其迅速，但这个速率很快变慢作为细胞扩散（面积和核间?...

Watch this Scientific Journal Video about Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing at JoVE.com

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

斑马鱼角膜基质细胞的表外植迁移，并提供了一个体外模型集体细胞迁移的上皮伤口愈合的情况下机制的研究。这些协议的细节，以建立初级外植体培养在集体细胞迁移测定法使用的有效方式。

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Research

JoVE Journal

Developmental Biology

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

内皮细胞迁移评估暴露于有毒化学品后，

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

科研

发育生物学

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

伤口划痕试验的优化来探测在小鼠间充质干细胞迁移的可溶性香烟烟雾提取物受损后

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

方法在成纤维细胞迁移的规律研究大分子复合物含MRP4，

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

腺病毒基因治疗对糖尿病性角膜病变：对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

一种在96孔板表演细胞迁移/伤口愈合设备

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...