Name: generation of organotypic raft cultures from primary human keratinocytes
Uploaded: 2012-02-22T13:00:00
Description: Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

作者要感谢萨莉·罗伯茨（英国伯明翰大学，英国伯明翰）的那种的E1E4抗体的礼物。这项工作是由国家癌症研究所（4R00CA137160-03）的赠款支持。

1。器官筏文化的制备<ol><li>金属筏电网的先处理，以消除任何可能干扰与分化过程中的残留铬硫酸。一小时含硫酸的玻璃烧杯中浸泡的金属网格，然后用自来水不断冲洗过夜。筏电网隔夜冲洗后，应为3-5小时，在双蒸水冲洗。 </li><li>提供支持排上，弯曲筏约0.5厘米的距离相等，彼此的三个方面。金属网格，然后进行高压灭菌。 </li><li>准备加入2.2克NaHCO3和4.8ĞHEPES的10X重组缓冲区。溶解在100毫升0...

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	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

我们在这里介绍一个可以用来研究一般上皮细胞分化的方法，但也可以很容易地适应研究病毒的发病机制，以及潜在疗法的疗效。许多病毒的目标上皮细胞感染的主站点，在人乳头状瘤病毒的情况下，或在一些点在病毒的生命周期，与疱疹病毒。虽然越来越多器官筏文化是费时，忠实地在体内的上皮细胞分化概括提供了一个非常有用的方法来检查病毒：宿主细胞相互作用的能力。成功地培?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>鼠尾胶原1型（4-5mg/ml） </td><td> BD公司</td><td> 354236 </td></tr><tr><td>没有NaHC0 3 DMEM培养液</td><td> Invitrogen公司</td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td>西格玛</td><td> S5761 </td></tr><tr><td> HEPES </td><td> Calbiochem公司</td><td> 391338 </td></tr><tr><td>不锈钢金属网格</td><td>威廉姆斯和秉性有限公司</td><td> CR-03063-040-100-S的</td></tr><tr><td> Ë中等11 </td><td></td><td></td></tr><tr><td>表皮生长因子</td><td> BD公司</td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

器官的上皮筏子文化的发展提供了一个有效的研究人员在体外系统，忠实地概括上皮细胞分化。这个系统有许多用途。例如，增长角质细胞的三维器官筏文化的能力一直在研究人类乳头状瘤病毒（HPV）的1重要里程碑。人乳头状瘤病毒的生命周期是紧密相连的鳞状上皮分化2。器官的上皮筏子文化展示在这里重现整个乳头状瘤病毒的生命周期，包括病毒生产3,4,5。此外，这些竹筏文化表现不典型增生病变相似， 在体内与HPV感染后观察。因此，该系统也可用于研究上皮细胞癌，以及药物对一般的上皮细胞分化的影响。最初由Asselineau和Prunieras开发<s高达6&gt;和高班等 7修改，器官上皮筏式养殖系统已经成熟到一般，比较容易培养模型，其中包括对胶原细胞的生长，插头保持在气-液界面（图1A）。在10-14天的过程中，细胞分层和分化，形成一个完整的厚度的上皮细胞，产生特定的分化，细胞角蛋白。收获筏可组织学检查，以及标准的分子生物学和生物化学技术。在这篇文章中，我们描述了从初级人角质形成细胞的生成筏文化的方法。可以用同样的技术，建立上皮细胞株，并可以很容易地适应与利用上皮组织正常或病变的活检8。许多病毒目标或者其复制的生命周期的一部分的皮肤或粘膜上皮。在过去的几年中，使用的可行性器官筏邪教作为一个研究病毒宿主细胞相互作用的方法URES已经证明以及几个疱疹病毒，腺病毒，细小病毒，痘病毒9。因此可以适应器官筏文化研究病毒的发病机制，并测试新的抗病毒药物，这些病毒是不是永久细胞株耕地的唯一手段。

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

一个<em&gt;在体外</em&gt;方法来模仿<em&gt;在体内</em&gt;上皮细胞分化的描述。许多病毒针对病毒生命周期的一部分上皮细胞，这种方法提供了一种研究病毒的手段：宿主的相互作用，更接近于，<em&gt;在体内</em&gt;。这种技术可用于小学的角质细胞，建立细胞系，以及正常或病变的活检组织。

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Research

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Immunology and Infection

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

科研

免疫与感染

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Preparation and Applications of Organotypic Thymic Slice Cultures

制备及胸腺器官切片文化中的应用

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

小RNA转染原代人Th17细胞通过下一代电穿孔

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...