转基因器官皮肤培养使用灭活人真皮代
Summary
The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.
Abstract
器官型培养允许它模仿功能和其在体内的组织对应的生理3D环境进行细胞-细胞接触和细胞-基质相互作用至关重要的重构。这是通过皮肤器官培养忠实地概括了表皮分化和分层方案例证。原代人表皮角质形成细胞是通过逆转录病毒基因在那里可以很容易地过度表达或撞倒的基因操纵。这些遗传修饰的角质形成细胞随后可以用来再生人表皮在器官型培养物的皮肤提供了强大的模型来研究遗传途径冲击表皮生长，分化和疾病进展。这里介绍的协议描述方法以制备灭活人真皮以及遗传操作初级人角质形成细胞，以便产生皮肤器官培养物。再生人体皮肤可downstr使用EAM的应用，如基因表达分析，免疫染色，和染色质免疫沉淀，随后通过高通量测序。因此，这些代转基因器官皮肤培养物将允许基因是保持皮肤稳态的关键的确定。
Introduction
人类表皮是一个分层上皮，它可通过细胞外基质被称为基底膜zone.The表皮不仅作为不可渗透的屏障，以防止水分的损失，而且作为防御的第一道防线，以保护连接到下面的真皮身体从国外和有毒物质1。基底层，这是表皮的最深处层，包含表皮干细胞和祖细胞的产生的已分化后代，形成了表皮2的其余部分。表皮祖细胞分化它们迁移向上，以形成被称为棘层3分化的细胞的第一层。在棘层，细胞打开角蛋白1和10的表达，其然后提供强度来承受物理应力为表皮的分化层。作为棘层细胞进一步分化，它们向上移动在表皮为米颗粒层其特征是，形成的透明角质和片状颗粒以及其组装下面质膜结构蛋白。随着细胞继续在分化过程中，质膜下方的蛋白质交联到对方，而层状颗粒从细胞挤出 ，以形成富含脂质屏障称为角质层4。
疾病涉及在表皮生长和分化的影响〜人口5的20％的改变。因此，了解该方法的机制是非常重要的。因为许多这些疾病的表现是在细胞-细胞或细胞-基质接触队伍，器官型培养其中人表皮复原在3D环境中已创建6-10。这些方法通常包括使用接种在细胞外基质的主要或转化角化细胞如失活的人类的真皮层，基质胶或胶原蛋白。
要明白，在表皮生长和分化的重要的基因调控机制，角化细胞可以遗传通过逆转录病毒载体操纵以拦截或过表达的基因在二维培养，然后在三维重构。这些方法已被广泛用于表征涉及表皮干细胞和祖细胞的自我更新和分化以及进展为肿瘤11-21基因。这里，提供了深入协议，关于如何通过利用逆转录病毒的改变在表皮器官型培养的基因表达。
Protocol
根据加州大学圣迭戈分校的研究伦理委员会的指导方针进行了人体皮肤的协议。人的皮肤可以从废弃的手术样本中获得，或从皮肤银行购买（皮肤银行被列在材料/设备表 ）。其中皮肤是从主或施主的年龄衍生的位置不是本实验只要基底膜区蛋白（胶原蛋白/层粘连蛋白）在真皮临界不会降解。 1.准备失活的人真皮的在接收到人体皮肤时，将组织在组织培?…
Representative Results
在产生器官型人皮肤的第一步是从真皮除去表皮。皮肤在37℃下在4×青霉素/链霉素/ PBS两周孵化应该允许真皮从表皮（图1A）的分离。如果分离表皮和真皮层，很难然后将组织在37℃于4笔/链球菌/ PBS一个星期，然后再次尝试剥离使用镊子。 一个关键的灭活真皮再生人表皮的是，确保了角质形成细胞被放置在真皮的正确的一面。真皮具有顶部和底部。真皮的顶部是?…
Discussion
遗传操作在人皮肤器官型培养提供许多优点常用研究2D培养细胞以及小鼠模型。 2D培养缺乏完整的组织和器官中发现的三维细胞 – 细胞和细胞 – 细胞外基质相互作用。最近的研究还发现，与三维培养的细胞显示多基因表达的相似性对原代人皮肤肿瘤16的二维和三维培养的皮肤癌细胞之间的巨大差异。人体器官皮肤的文化也有过小鼠模型的几个优点。小鼠模型是耗时且昂贵的产生以及人类和?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是supportedby美国癌症协会的研究学者格兰特（RSG-12-148-01-DDC）和CIRM基础生物学奖（RB4-05779）。
Materials
| Human skin | New York Firefighters Skin Bank | http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html |
| PEN/STREP | GIBCO | 15140-122 |
| amphotropic phoenix cell lines | ATCC | CRL-3213 |
| FUGENE 6 transfection reagent | Promega | E2691 |
| Keratinocyte Media (KCSFM) | Life Technologies | 17005042 |
| DMEM | GIBCO | 11995 |
| Ham's F12 | Cambrex | 12-615F |
| FBS | GIBCO | 10437-028 |
| Adenine | Sigma | A-9795 |
| Cholera Toxin | Sigma | C-8052 |
| Hydrocortisone | Calbiochem | 3896 |
| Insulin | Sigma | I-1882 |
| EGF | Invitrogen | 13247-051 |
| Transferrin | Sigma | T-0665 |
| Ciprofloxacin Hydrochloride | Serologicals | 89-001-1 |
| cautery | Bovie Medical Corporation | AA01 |
| Matrigel | Corning | 354234 |
| Keratin 1 antibody | Biolegend | PRB-149P |
| square pegs | Arts and crafts stores | |
| human neonatal keratinocytes | ATCC | PCS-200-010 |
| human neonatal keratinocytes | Cell Applications | 102K-05n |
| MSCV retroviral vector | Clontech | 634401 |
| LZRS retroviral vector | Addgene | |
| pSuper.Retro.Puro Retroviral vector | Oligoengine | VEC-PRT-0002 |
| hexadimethrine bromide | Sigma | H9268-5G |
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Citer Cet Article
Li, J., Sen, G. L. Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis. J. Vis. Exp. (106), e53280, doi:10.3791/53280 (2015).