Name: experimental generation of carcinoma-associated fibroblasts (cafs) from human mammary fibroblasts
Uploaded: 2011-10-25T12:00:00
Description: Watch this Scientific Journal Video about Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts at JoVE.com

我们感谢慷慨支持和监督这项工作，并为这篇稿子的关键编辑基兰Mellody先生（英国曼彻斯特大学，曼彻斯特）罗伯特A ·温伯格博士（怀特黑德生物医学研究，剑桥研究所）。这个项目得到了英国研究（CR英国）授予数量C147/A6058（AO）

1。原代培养人正常乳腺成纤维细胞的分离隔离原代培养人正常乳腺成纤维细胞的实验程序概述图。 1A。 <ol><li>准备的细胞分解缓冲区，作为描述以前12：10％胎儿小牛血清（FCS），青霉素，链霉素（200单位/毫升），胶原酶型我（1毫克/毫升）和透明质酸（贝科的修改鹰的中等（DMEM） 125单位/ ml）。 </li><li>多次在磷酸盐缓冲液（PBS）从减少隆乳（〜0.5克）洗净乳房组?...

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	<li>Ronnov-Jessen, L., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+by+proxy:+can+the+microenvironment+be+both+the+cause+and+consequence?.">Breast cancer by proxy: can the microenvironment be both the cause and consequence?.</a> Trends Mol. Med. 15, 5-13 (2009).</li><li>Mueller, M. M., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friends+or+foes+-+bipolar+effects+of+the+tumour+stroma+in+cancer.">Friends or foes - bipolar effects of the tumour stroma in cancer.</a> Nat. Rev. Cancer. 4, 839-849 (2004).</li><li>Bhowmick, N. A., Neilson, E. G., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer+initiation+and+progression.">Stromal fibroblasts in cancer initiation and progression.</a> Nature. 432, 332-337 (2004).</li><li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+cancer.">Fibroblasts in cancer.</a> Nat. Rev. Cancer. 6, 392-401 (2006).</li><li>De Wever, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenascin-C+and+SF/HGF+produced+by+myofibroblasts+in+vitro+provide+convergent+pro-invasive+signals+to+human+colon+cancer+cells+through+RhoA+and+Rac.">Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac.</a> 18, 1016-1018 (2004).</li><li>Serini, G., Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+myofibroblast+activity+and+phenotypic+modulation.">Mechanisms of myofibroblast activity and phenotypic modulation.</a> Exp. Cell. Res. 250, 273-283 (1999).</li><li>Shimoda, M., Mellody, K. T., Orimo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+are+a+rate-limiting+determinant+for+tumour+progression.">Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression.</a> Semin. Cell. Dev. Biol. 21, 19-25 (2010).</li><li>Pietras, K., Ostman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+interactions+with+the+tumor+stroma.">Hallmarks of cancer: interactions with the tumor stroma.</a> Exp. Cell. Res. 316, 1324-1331 (2010).</li><li>Polyak, K., Haviv, I., Campbell, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-evolution+of+tumor+cells+and+their+microenvironment.">Co-evolution of tumor cells and their microenvironment.</a> Trends in Genetics. 25, 30-38 (2009).</li><li>Franco, O. E., Shaw, A. K., Strand, D. W., Hayward, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+associated+fibroblasts+in+cancer+pathogenesis.">Cancer associated fibroblasts in cancer pathogenesis.</a> Semin. Cell. Dev. Biol. 21, 33-39 (2010).</li><li>Kojima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+TGF-beta+and+stromal+cell-derived+factor-1+(SDF-1)+signaling+drives+the+evolution+of+tumor-promoting+mammary+stromal+myofibroblasts.">Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 20009-20014 (2010).</li><li>Orimo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+present+in+invasive+human+breast+carcinomas+promote+tumor+growth+and+angiogenesis+through+elevated+SDF-1/CXCL12+secretion.">Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.</a> Cell. 121, 335-348 (2005).</li></ol>

非洲足联特定的标记和异质性水平缺乏观察，其中包括水产科学研究院提供的这种类型的细胞的特性本身就是一个挑战。 在体外研究水产科学研究院也已阻碍了新的复杂情况，这些细胞衰老停止增殖，长期培养。我们以前尝试直接immortalise使用端粒酶逆转录病毒基因构造的主要水产科学研究院是不成功的。因此，要进一步研究肿瘤促进这些细胞的特性，我们开发了一种方法来生成使用coimpla...

<table border="1"><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液</td><td></td><td> Invitrogen公司</td><td> 61965-026 </td><td></td></tr><tr><td>胎牛血清</td><td></td><td> GIBCO公司</td><td> 10270 </td><td></td></tr><tr><td>青霉素链霉素</td><td></td><td> Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td>胶原酶I型</td><td></td><td>西格玛</td><td> C0130 - 1G </td><td></td></tr><tr><td>透明质酸</td><td></td><td>西格玛</td><td> H4272 </td><td></td></tr><tr><td>波形蛋白（V9）抗体</td><td></td><td> Novocastra实验室</td><td> NCL - L - VIM - V9 </td><td></td></tr><tr><td>腱糖蛋白C（BC - 8）抗体</td><td></td><td></td><td></td><td>从礼物 博士<stro吴&gt;卢西亚诺 Zardi </td></tr><tr><td> α- SMA - Cy3标记抗体（1A4） </td><td></td><td>西格玛</td><td> C6198 </td><td></td></tr><tr><td>脯氨酰- 4 -羟化酶</td><td></td><td> Dako公司</td><td> M0877 </td><td></td></tr><tr><td> （5B5）抗体</td><td></td><td></td><td></td><td></td></tr><tr><td>胶原TYPE1 1A抗体</td><td></td><td>西格玛</td><td> HPA011795 </td><td></td></tr><tr><td>泛角蛋白抗体</td><td></td><td>西格玛</td><td> C5992 </td><td></td></tr><tr><td>纤维连接蛋白抗体</td><td></td><td> BD公司</td><td> 610077 </td><td></td></tr><tr><td> S100A4/FSP-1（成纤维细胞特定的蛋白质- 1）抗体</td><td></td><td> Dako公司</td><td> A5114 </td><td></td></tr><tr><td>成纤维细胞表面蛋白 （克隆1B10）抗体</td><td></td><td> Abcam公司</td><td> ab11333 </td><td></td> &lt;/ TR&gt; <tr><td> MSCV - IRES - GFP的构建</td><td></td><td></td><td></td><td>请求的作者</td></tr><tr><td> pBabe - PURO兴建</td><td></td><td></td><td></td><td>购买从Addgene </td></tr><tr><td>嘌呤霉素</td><td></td><td>西格玛</td><td> P8833 </td><td></td></tr><tr><td> DAPI </td><td></td><td>西格玛</td><td> D9564 </td><td></td></tr><tr><td> 15毫升锥形管</td><td></td><td>康宁</td><td> 430766 </td><td></td></tr><tr><td>裸鼠</td><td></td><td>泰康利</td><td> NCRNU - F </td><td>女性NCR裸体</td></tr><tr><td> C3H/10T1/2细胞</td><td></td><td> ATCC </td><td></td><td> CCL - 226 </td></tr></table>

experimental generation of carcinoma-associated fibroblasts (cafs) from human mammary fibroblasts

癌肿瘤细胞和非癌变的车厢称为“基质”组成的复杂的组织。基质由细胞外基质（ECM）和各种间质细胞，包括成纤维细胞，肌纤维母细胞，内皮细胞，周细胞和白细胞1-3。 肿瘤相关基质是邻近的肿瘤细胞释放大量的旁分泌信号作出反应。在疾病的过程中，基质往往成为人口，包括大量的肌纤维母细胞癌相关成纤维细胞（CAFS）。这些细胞先前已提取他们在体外培养来自许多不同类型的人类癌的。一个亚群水产科学研究院是通过上调α-平滑肌肌动蛋白（α- SMA）的表达4,5区别。这些细胞是一种“激活成纤维细胞”的标志，与肌纤维母细胞彗星类似物业ommonly观察受伤和纤维化组织6。这种肌纤维母细胞非洲足联子集的存在是高度相关，高品位的恶性肿瘤，并与患者的不良预后相关。 许多实验室，包括我们自己，已经表明，咖啡馆，当肿瘤细胞注射到免疫缺陷小鼠，能够大大促进tumourigenesis 7-10。然而，从癌患者准备的咖啡馆，经常接受各地正在进行的实验使用的广泛性，传播文化的限制期间衰老。为了克服这个困难，我们开发了一种新技术，对实验产生永生非洲足联的人类乳腺细胞株人乳腺成纤维细胞（EXP - CAFS），使​​用coimplantation乳腺肿瘤异种移植模型。 为了生成EXP -水产科学研究院，父母的人乳腺成纤维细胞，减少隆乳组织获得，分别为第一immortaliSED与端粒酶，端粒酶全酶的催化亚基，并表达GFP和嘌呤霉素抗性基因工程。 coimplanted与MCF - 7人乳腺癌表达小鼠异种移植成一个激活的ras癌基因（MCF - 7 - ras基因细胞）的细胞，这些细胞。最初注射的人乳腺成纤维细胞体内孵化期后，分别提取其嘌呤霉素抗性11的基础上，在肿瘤异种移植。 我们观察到，居民人乳腺成纤维细胞分化，采用肌纤维母细胞表型和后天在肿瘤进展过程中促进肿瘤的性质。重要的是，这些细胞，定义EXP - CAFS，密切模仿从乳腺癌患者解剖隔离CAFS的肿瘤促进肌纤维母细胞表型。因此，我们的肿瘤异种移植派生EXP - CAFS提供一个有效的模型来研究人类breas的生物学水产科学研究院吨癌。所描述的协议也可以用于生成和表征各种非洲足联衍生出其他类型的人类癌的人群扩展。

Watch this Scientific Journal Video about Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts at JoVE.com

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

癌相关成纤维细胞（CAFS）在肌纤维母细胞肿瘤间质内的丰富，推动肿瘤进展发挥了重要作用。从人类乳腺成纤维细胞的实验产生CAFS我们开发了一个coimplantation肿瘤xengraft模型。协议描述了如何建立非洲足联的肌纤维母细胞的获取能力，以促进tumourigenesis。

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

科研

医学

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

人乳内动脉（IMA）移植及内支架置入术：人体模型支架内再狭窄的研究开发

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

推导人类纤维原细胞的皮肤穿刺活检植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Experimental Human Pneumococcal Carriage

人体实验肺炎球菌运输

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

An Ex vivo Model to Study Hormone Action in the Human Breast

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

后皮肤微移植生成一个新的方法解聚表征的组织

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...