We thank Dr. Tadashi Koyama for technical help. This work was supported in part by JSPS KAKENHI Grant Numbers 23249045, 15K09211, 15K19172; a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan; a grant for research on allergic disease and immunology, Japan.

1.准备肺上皮细胞的培养和<ol><li>培养A549人肺上皮细胞（贴壁细胞系）中的Dulbecco改进的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），100IU / ml青霉素，和100μg/ ml链霉素。 </li><li>卸下并丢弃从培养皿的细胞培养基，并用5洗一次 - 10毫升磷酸盐缓冲盐水（PBS）中。清洗后，立即吸出PBS。 </li><li>加2ml胰蛋白酶/乙二胺四乙酸（EDTA）（0.05％），在37℃下孵育，5％CO 2的3分钟。 </li><li>收集脱离的细胞在含有细胞培养基，离心在RT管在150×g下4分钟离心管中。 </li><li>悬浮细胞在2毫升细胞培养基的沉淀并除去对细胞计数的样品。算使用台盼蓝染色血球以检查细胞存活力细胞的数目。 </li><li>种子A549细胞上10厘米聚苯乙烯板在0.5的密度- 1.0×10 6细胞/皿含10毫升凝胶收缩测定培养基的。种子上6孔聚苯乙烯板的细胞以0.5的密度- 1.0×10 5个细胞/孔用2ml为EMT确认培养基。孵育细胞在37℃和5％CO 2的24小时。 </li></ol> 2. EMT过程<ol><li>添加TGF-β1（5微克/毫升）的10微升和10微升的TNF-α（10微克/毫升），以在步骤1.6接种的板凝胶收缩试验。添加TGF-β1（5微克/毫升）的2微升和2μl的TNF-α（10微克/毫升）至板在步骤1.6接种EMT确认。在37℃和5％CO 2的48小时。 </li></ol> 3.确认EMT过程的PCR和免疫印迹<ol><li>确认形态变化（由鹅卵石状，以纺锤形）用相差显微镜处理的细胞中。 注:规范人A549细胞有鹅卵石状和三角形的外观是上皮细胞的特征，但与TGF-β1和肿瘤坏死因子α刺激后，细胞出现长和梭形是类似于间充质细胞14,15。 </li><li>评估上皮标记物，如E-钙粘蛋白的表达，和间充质标记物如N-钙粘蛋白，波形蛋白和α平滑肌肌动蛋白使用PCR或印迹。 <ol><li>使用RNA提取试剂盒16中的细胞中提取总RNA，并使用根据制造商的协议逆转录17合成cDNA。 </li><li>测量使用实时PCR系统18，并根据制造商的说明单体花青染料PCR试 ​​剂盒的mRNA水平的表达。对于GAPDH特异性引物（甘油醛-3-磷酸脱氢酶），ACTA2（的α-肌动蛋白2），CDH1（钙粘蛋白-1; E-钙粘蛋白），和VIM（波形蛋白）示于表1中 </st荣&gt;。 </li><li>裂解用裂解缓冲液（ 表2）含有1％蛋白酶抑制剂混合物溶液中的细胞。测量使用蛋白测定试剂盒19,20的所有样品的蛋白浓度，并应用相同量的蛋白质，以聚丙烯酰胺凝胶。 <ol><li>执行SDS凝胶电泳和蛋白质的半干转移到PVDF膜21。 2小时 - 在1封闭缓冲液孵育初级抗体的膜。温育后，用洗涤缓冲液洗两次膜孵育与第二抗体在膜1小时。 注:请参阅材料/设备表在这些研究中使用的抗体和稀释。 见表2为封闭缓冲液的组分。 </li><li>两次洗涤用洗涤缓冲液的膜。就拿膜的照片与使用冷CCD相机11,23蛋白质印迹检测试剂盒22。 </li></ol></li></ol></li></ol> 4。凝胶收缩试验评定EMT <ol><li>吸从细胞培养容器中的条件培养基，并用5洗井 - 10mL的PBS，以去除死细胞。清洗后，立即吸出PBS。 </li><li>加2ml的0.05％胰蛋白酶/ EDTA中，并在37℃反应和5％的CO 2 3分钟。 </li><li>收集脱离的细胞在含有DMEM补充有蛋白酶抑制剂（1毫克/毫升），离心管在室温下在150×g下4分钟离心管中。 </li><li>混合型胶原凝胶用蒸馏水，和4×浓缩的DMEM以调节体积以达到1.75毫克/毫升的胶原浓度，和1×DMEM的浓度。一定要保持冰凝胶培养基在这一步。 注意:为了让6毫升凝胶培养基中，拌匀3.5毫升1型胶原凝胶（3毫克/毫升），1.5毫升4×浓的DMEM和1 ml蒸馏水。 </li><li>悬浮于500μlPBS中的细胞沉淀并除去对细胞计数的样品。计数使用血球用锥虫蓝染色的细胞的数目来检查细胞存活力。 </li><li>添加凝胶介质来调整卷来实现/孔3.0×10 5个细胞的细胞密度（6.0×10 5个细胞/ ml），轻轻地但很快没有凝胶化，通过移液混合。 </li><li>分配将0.5ml混合物成24孔未处理的板的每个孔迅速并小心地使整齐圆筒形。要小心，不要让任何气泡污染凝胶（ 图1A）。 注意:含有细胞的凝胶介质是粘性的，并且可以很容易地在井胶凝并形成月牙形状。 </li><li>在37℃下孵育在细胞培养箱15分钟的盘，5％CO 2和95％的湿度完全胶凝。 </li><li>分离从板凝胶而不通过的方式移动的无菌刮刀在一个方向上绘制的圆周断裂。用刮刀轻轻转移凝胶至60mm组织培养皿含有5毫升含或不含TGF-β1（5毫微克/毫升）和TNF-α（10毫微克/毫升）的DMEM / 1％FBS的。 </li><li>轻轻摇动菜，以确保凝胶漂浮在介质上。在37℃，5％CO 2和95％湿度下孵育在细胞培养箱中。 </li></ol> 5.凝胶尺寸的测量<ol><li>后使用图象分析系统0，24，48，和72小时测量胶原蛋白凝胶的尺寸。 <ol><li>打开凝胶文档系统（ 图2A），并把餐具放入光屏蔽柜。然后起飞的内阁菜盖子。 </li><li>打开相关的凝胶分析软件。单击菜单栏中的"图像采集按钮"，显示在机柜凝胶的形象。然后，点击"获取"，在菜单栏取凝胶（ 图2B）的照片。 </li><li>单击菜单栏（ 图2C）中的"检测键"，调整测量区域（黄圈我Q 图2C）通过拖动鼠标。然后，单击菜单栏中的"自动检测按钮"（ 见图2D按钮）。该软件会自动检测出热图（ 图2D）凝胶。 </li><li>点击"确定"，以提取图像处理凝胶的轮廓和计算的轮廓（ 图2E）所包围的区域。的区域被计算后，将计算出的区域被显示在单独的弹出窗口。 注意:如果凝胶成像期间彼此重叠，轻轻用无菌移液管尖移到凝胶。 </li></ol></li></ol>

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<li>Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+cytokines+augments+TGF-beta1-induced+epithelial-mesenchymal+transition+in+A549+cells+by+up-regulating+TbetaR-I%5BTitle%5D)+AND+%22Cell+motility+and+the+cytoskeleton%22%5BJournal%5D)">Inflammatory cytokines augments TGF-beta1-induced epithelial-mesenchymal transition in A549 cells by up-regulating TbetaR-I.</a> Cell motility and the cytoskeleton. 65, 935-944 (2008).</li>
</ol>

作者有没有利益冲突披露。

在这项研究中开发的协议包括两个步骤。进行第一步骤以诱导EMT的，而第二个步骤是在凝胶收缩试验。因为它确认细胞发生EMT是很重要的，第2步提供了一个极好的补充，形态学和基因表达的变化。以往的研究表明，A549细胞的EMT诱导仅24，TGF-β1;然而，正如我们以前10日报道，TNF-α处理增强EMT和收购的间充质细胞标志物。它被认为是TGF-β介导EMT的机制是SMAD依赖性25。 TNFα增强了TGF-β1诱导的EMT，导致增强凝胶收缩。已经报道了TNFα的用于增强TGF-β1诱导的EMT的机制不仅Smad2的接头区的磷酸化，但也MAPK信号调节26。因此，在该研究开发的协议包括stimul由两个TGF-β1和TNF-α的通货膨胀。 该行的EMT到I型胶原凝胶的类型，和浮动在介质上的细胞的嵌入，是该测定（步骤4）的中央的方面。由于凝胶是软的，容易损坏，并且在每侧有不同的尺寸，施加凝胶时向培养基中并测量它们的尺寸格外小心是必需的。如果难以进行这种步骤中，是测量井的凝胶尺寸而不凝胶移入60mm培养皿27,28的替代方法。有几个共同的问题，第一个问题是，胶原凝胶容易凝胶化在RT，所以凝胶如在步骤4.7中提到经常变形。因此，该板块一定要轻轻地铸造成凝胶井，以确保他们采取一个整洁的圆柱状后动摇。第二个问题是，如果任何气泡凝胶化期间输入的凝胶，然后将凝胶的大小将是不均匀的。要小心，不要让任何空气bubbl下载到污染凝胶。有原始的方法，这方法之间有两个主要差异。第一差别是该原始方法的胶原凝胶的最终浓度为0.75毫克/毫升，但是，该方法的是1.75毫克/毫升，以使凝胶收缩超过他们做的原始方法。第二差异是，原来的方法使用凝胶浮动平台无血清培养基，但这种方法中，以保持细胞生存力和维持细胞收缩使用1％FBS中的凝胶的浮动平台。 最初为成纤维细胞开发的凝胶收缩试验是在体外模型用于评估有助于伤口愈合和纤维化13的过程的细胞的收缩性的理想。成纤维细胞的1型胶原的连接应该是产生机械张力该因而导致在胶原凝胶的尺寸的减小。此外，该测定中，最近已经使用作为研究的炎性疾病，包括支气管哮喘和COPD 29-31气道细胞的收缩的体外模型。 凝胶收缩试验是可比其他测定法，诸如细胞迁移测定的，因为它不需要特定设备和显示出高的定量重复性。但是，也有施加凝胶收缩试验用于评估EMT 32报道很少。在这项研究中，包含该行的EMT的细胞凝胶显著在大小减小24到72小时的凝胶化后，指示细胞收缩。然而有这种方法的局限性。第一个是，在该测定中，凝胶的尺寸变化显示在凝胶所有细胞的收缩力的总和。然而，这是难以评价在使用该测定的凝胶单细胞收缩。第二个是，A549细胞是癌症细胞系，而不是正常的人上皮细胞。因此，它是难以推断ö乌尔结果直接肺纤维化的发病机制。然而，A549细胞被广泛用于研究呼吸道上皮细胞的功能，并且一些研究通过使用A549细胞肺纤维化33,34的模型得到的EMT的证据。 总之，I型胶原蛋白包含经历EMT的大小减少了，指示这些细胞的收缩间充质细胞的凝胶。因此，凝胶收缩试验应被视为一个扩展体外测定中通过EMT过程中的细胞，以评估采集收缩功能。

纤维化参与多种慢性进行性疾病，如间质性肺疾病，心脏纤维化，肝硬化，终端肾功能衰竭，系统性硬化，和自身免疫性疾病1的发病机制。间间质性肺疾病，特发性肺纤维化（IPF）是一种慢性进行性疾病，并显示预后差。 IPF的病理特点是成纤维细胞灶组成了与预后有关激活成纤维细胞和肌的发展。提出这样的肺成纤维细胞的起源是从几个间充质细胞，包括原驻地肺成纤维细胞和骨髓循环纤维细胞衍生的。最近，上皮-间质转化（EMT）已经提出了用间充质细胞2的形成相关联，并且有助于纤维化疾病的发病机制。 它被认为是电磁ŧ在胚胎发育过程中的重要作用，伤口愈合和癌症的进展，包括肿瘤侵袭和转移3。以下EMT的过程中，上皮细胞的上皮标记物，如E-钙粘蛋白的损失获得间充质细胞的能力，以及由间充质标志物，如波形蛋白和α平滑肌肌动蛋白（SMA）4,5的表达。以往的研究表明，EMT过程已与组织纤维化的发展在肾脏6和肺7被相关联的证据。此外，慢性炎症促进纤维化疾病8;此外，如炎性细胞因子如肿瘤坏死因子超家族成员14（TNFSF14; LIGHT），肿瘤坏死因子（TNF）-α和白细胞介素1β，已显示出提高的EMT 9-12。 胶原凝胶收缩试验，其中成纤维细胞嵌入型我一个基于胶原的细胞收缩测定胶原凝胶三维， 在体外模型收缩的评价理想。收缩是成纤维细胞的特征功能之一，并有助于正常的伤口修复和纤维化13。在该测定中，可以认为，成纤维细胞附着于I型通过整合素依赖性机制胶原被认为在一定条件下，以产生机械张力，并因此导致组织收缩。 这里，凝胶收缩测定的发展报告可以适于评估在经历EMT的细胞中采集收缩功能。这份报告表明，这种改进法是适用于评估在经历了EMT间质细胞收缩。

<table><tbody><tr><td>DMEM</td><td>sigma aldrich</td><td>11965-092</td><td>For A549 medium</td></tr><tr><td>FBS</td><td>GIBCO</td><td>10437</td><td></td></tr><tr><td>Transforming Growth Factor-&amp;beta;1, Human, recombinant</td><td>Wako Laboratory chemicals</td><td>209-16544</td><td></td></tr><tr><td>Recombinant Human TNF-&amp;alpha;</td><td>R&amp;amp;D systems</td><td>210-TA/CF</td><td></td></tr><tr><td>E-Cadherin (24E10) Rabbit mAb</td><td>Cell Signaling Technology</td><td>#3195</td><td>1:3,000 dilution</td></tr><tr><td>Vimentin (D21H3) Rabbit mAb</td><td>Cell Signaling Technology</td><td>#5741</td><td>1:3,000 dilution</td></tr><tr><td>Anti-&amp;alpha;-Tubulin antibody</td><td>sigma aldrich</td><td>T9026</td><td>1:10,000 dilution</td></tr><tr><td>Monoclonal Anti-Actin, &amp;alpha;-Smooth Muscle antibody&amp;nbsp;</td><td>sigma aldrich</td><td>A5228</td><td>1:10,000 dilution</td></tr><tr><td>Anti-N-cadherin antibody</td><td>BD Transduction Laboratories</td><td>#610920</td><td>1:1,000 dilution</td></tr><tr><td>Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)</td><td>GE Healthcare</td><td>NA931-100UL</td><td>1:20,000 dilution</td></tr><tr><td>Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)</td><td>GE Healthcare</td><td>NA934-100UL</td><td>1:20,000 dilution</td></tr><tr><td>Blocking reagent</td><td>GE Healthcare</td><td>RPN418</td><td>2% in TBS-T</td></tr><tr><td>6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid</td><td>corning</td><td>#353046</td><td></td></tr><tr><td>100 mm Cell culture dish</td><td>TPP</td><td>#93100</td><td></td></tr><tr><td>DMEM, powder</td><td>life technologies</td><td>12100-046</td><td>For 4&amp;times; DMEM</td></tr><tr><td>Type 1 collagen gel</td><td>Nitta gelatin</td><td>Cellmatrix type I-A</td><td></td></tr><tr><td>24 Well cell culture plate</td><td>AGC TECHNO GLASS</td><td>1820-024</td><td></td></tr><tr><td>Gel Documentation System&amp;nbsp;</td><td>ATTO</td><td>AE-6911FXN</td><td>Gel imager</td></tr><tr><td>Gel analyzing software</td><td>ATTO</td><td>Densitograph, ver. 3.00</td><td>analysing software bundled with AE-6911FXN</td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>life technologies</td><td>25300054</td><td></td></tr><tr><td>24 Well Plates, Non-Treated</td><td>IWAKI</td><td>1820-024</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>life technologies</td><td>15250-061</td><td></td></tr><tr><td>RNA extraction kit</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>Reverse transcriptase</td><td>life technologies</td><td>18080044</td><td></td></tr><tr><td>Real time PCR system</td><td>Stratagene</td><td>Mx-3000P</td><td></td></tr><tr><td>SYBR green PCR kit</td><td>Qiagen</td><td>204145</td><td></td></tr><tr><td>Protease Inhibitor Cocktail (100x)</td><td>life technologies</td><td>78429</td><td></td></tr><tr><td>PVDF membrane</td><td>ATTO</td><td>2392390</td><td></td></tr><tr><td>Protein assay kit</td><td>bio-rad</td><td>5000006JA&amp;nbsp;</td><td></td></tr><tr><td>Polyacrylamide gel</td><td>ATTO</td><td>2331810</td><td></td></tr><tr><td>Western blotting detection reagent</td><td>GE Healthcare</td><td>RPN2232</td><td></td></tr><tr><td>Cold CCD camera</td><td>ATTO</td><td>Ez-Capture MG/ST</td><td></td></tr><tr><td>Trypsin inhibitor</td><td>sigma aldrich</td><td>T9003-100MG</td><td></td></tr><tr><td>Polyoxyethylene (20)Sorbitan Monolaurate</td><td>Wako Laboratory chemicals</td><td>163-11512</td><td></td></tr><tr><td>Polyoxyethylene (9) octyiphenyl ether</td><td>Wako Laboratory chemicals</td><td>141-08321</td><td></td></tr></tbody></table>

development of an in vitro assay to evaluate contractile function of mesenchymal cells that underwent epithelial-mesenchymal transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

EMT期间，上皮细胞失去上皮标记，如E-钙粘蛋白，并获得间充质标志物，如波形蛋白和α平滑肌肌动蛋白4,5的表达。与TGF-β1和肿瘤坏死因子αA549人肺上皮细胞的温育诱导EMT。正常A549细胞的外观是鹅卵石状的形状，三角形形状是上皮细胞（ 图3A）的特性，但是刺激的TGF-β1和TNF-α后，外观改变为长纺锤形是类似于间充质细胞（ 图3B）。 的上皮细胞和间质标记物的表达进行评价，确认细胞发生EMT。相对mRNA的表达与ΔΔCT法计算。个人数据被归一针对甘油醛-3-磷酸脱氢酶（GAPDH），它是一个内务克烯。与TGF-β1和肿瘤坏死因子α48小时处理的A549细胞已显著减少CDH1的表达和显著增加VIM和ACTA2（ 图4A）的表达。用于Q-PCR的引物序列示于表1。如在图4B中 ，刺激TGF-β1和肿瘤坏死因子α减毒E-钙粘蛋白的表达示出，而波形蛋白，N-钙粘蛋白，α平滑肌肌动蛋白的表达诱导。 进行凝胶收缩试验，以评估经历EMT的细胞的收缩性。诱导与TGF-β1和肿瘤坏死因子αEMT后，将细胞浇铸成胶原蛋白凝胶并孵育含有TGF-β1和TNF-α，或PBS 72小时中的介质。 如图5A所示，含有TGF-β1和TNF-α处理的细胞中的凝胶比含有用PBS处理的细胞控制凝胶小。要QUAntify在凝胶大小的变化，凝胶使用凝胶分析器0，24，48，和处理后72小时进行分析。 72小时后，含有TGF-β1和肿瘤坏死因子α处理的细胞凝胶的尺寸均显著减少与对照相比的凝胶（ 图5B）。我们还证实，含有TGF-β1处理的细胞的凝胶单独比对照凝胶小，但为（数据未显示）比TGF-β1和肿瘤坏死因子α处理的凝胶不小。 <img alt="图1" src="/files/ftp_upload/53974/53974fig1.jpg" /> 图1.补充图的制作凝胶。这些图像显示抛入井中的凝胶。含有细胞的凝胶介质是粘性的，并且可以容易胶凝并在井形式月牙形。 （A）左边的图像显示，该凝胶被正确铸造，右边的图片显示"整洁的圆筒形"的模式。 （B &lt;/强&gt;）左边的图像显示，该凝胶变形，并且右图像显示，气泡污染凝胶。 （C）的凝胶是软的，这样容易损坏，变形，通常像"吃豆人"， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53974/53974fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53974/53974fig2.jpg" /> 图2中的步骤测量的凝胶尺寸（A）的凝胶文档系统包括PC和凝胶成像系统。 （B）中的"图像获取按钮"，和凝胶的照片。 （C）中的"检测键"，和用于调整测量区域（黄色圆圈）的窗口。 （D）的"自动检测键"，并且该软件使得凝胶的热图从一个图象到脱<html> TECT凝胶的轮廓。 （E）该软件通过提取图像处理凝胶的轮廓，并计算出轮廓包围的地区。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53974/53974fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53974/53974fig3.jpg" /> 图3. EMT诱导细胞形貌。A549人肺上皮细胞以1.0×10 5个细胞/孔的密度在一个6孔板中铺板并有或没有5纳克/ ml的TGF-β1和10ng / ml的温育TNF-α为48小时，接着通过相差显微成像。 （A）和（B）是下×200所获得的图像。在（A）的比例尺和（B），100微米。<html> T ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53974/53974fig4.jpg" /> 图4. EMT刺激与TGF-β1和肿瘤坏死因子α。 （A）EMT标志物的表达（CDH1，VIM和 ACTA2）的定量RT-PCR分析。 （B）中示出E-钙粘蛋白，N-钙粘蛋白，波形蛋白的表达Western印迹，和α平滑肌肌动蛋白在有或无TGF-β1和TNF-α刺激的A549细胞的蛋白质。 α微管蛋白用作内部对照。 A549细胞分别为在图1中，N = 3次独立实验中培养。 ** P &lt;0.01;误差棒代表SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53974/53974fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 尔斯/ ftp_upload / 53974 / 53974fig5.jpg"/&gt; 图5.细胞发生EMT获得收缩 。在10 cm的培养皿1.0×10 6个细胞/皿的密度铺板并有或没有5纳克/ ml的TGF-β1和10ng / ml的TNF-α的48小时培养A549细胞。然后将细胞在24孔板铸造成500微升含有1.75毫克/毫升的胶原凝胶培养基以3.0×10 5个细胞/孔的密度。后的凝胶形成，它们被添加到培养基有或没有5纳克/ ml的TGF-β1和10ng / ml的60毫米培养皿中TNF-α和培养72小时，随后通过成像（A）中。凝胶尺寸被后使用图像分析系统0，24，48，和72小时进行测定。 N = 9个独立的实验（B）中。 ** P &lt;0.01;误差棒代表SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53974/53974fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:kEEP-together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 基因名称 </td><td colspan="2"> 前进5&#39;→3&#39; </td><td colspan="2"> 扭转5&#39;→3&#39; </td></tr><tr><td> ACTA2 </td><td colspan="2"> GCACCCAGCACCATGAAGA </td><td colspan="2"> ACCGATCCAGACAGAGTATTT </td></tr><tr><td> GAPDH </td><td colspan="2"> GGTGAAGGTCGGAGTCAACGGA </td><td colspan="2"> GAGGGATCTCGCTCCTGGAAGA </td></tr><tr><td> VIM </td><td colspan="2"> GACAATGCGTCTCTGGCACGTCTT </td><td colspan="2"> TTCTTCTGCCTCCTGCAGGTTCTT </td></tr><tr><td> CDH1 </td><td colspan="2"> CCCATCAGCTGCCCAGAAAATGAA </td><td colspan="2"> CTGTCACCTTCAGCCATCCTGTTT </td></tr></tbody></table> 表1引物序列。用于定量RT-PCR引物序列。 GAPDH用作内部对照。 <table border="1" fo:keep-together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 裂解液:RIPA缓冲液 </td><td></td></tr><tr><td>的20mM的Tris-HCl pH为7.5 </td><td></td></tr><tr><td> 150毫米氯化钠</td><td></td></tr><tr><td> 1毫摩尔EDTA </td><td></td></tr><tr><td> 1％聚氧乙烯（9）醚octyiphenyl </td><td></td></tr><tr><td> 0.1％的钠脱氧胆酸</td><td></td></tr><tr><td> 0.1％SDS中</td><td></td></tr><tr><td> 封闭液 </td><td></td></tr><tr><td>在洗涤缓冲液的1％封闭试剂</td><td></td></tr><tr><td>洗涤液:TBST缓冲液</td><td></td></tr><tr><td>氯化钠</td><td> 45克</td></tr><tr><td>的1M Tris pH 7.4的</td><td> 50毫升</td></tr><tr><td>聚氧乙烯（20）脱水山梨醇单月桂酸</td><td> 2.5毫升</td></tr><tr><td>水蒸馏</td><td>加入5升</td></tr><tr><td&gt;总</td><td> 5升</td></tr></tbody></table> 表2缓冲器的使用的组分。裂解的组分，封闭缓冲液和洗涤缓冲液。

Watch this Scientific Journal Video about Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition at JoVE.com

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

development-an-vitro-assay-to-evaluate-contractile-function

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

12.2K 次观看.  The University of Tokyo Hospital. Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

视频: Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜生长培养测定和<em&gt;离体</em&gt;心外膜源性细胞迁移的评估

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

科研

发育生物学

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

微型硅悬臂梁的利用率，评估细胞收缩功能<em&gt;在体外</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

生物工程

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

生物学

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

使用微图案弹性体对单细胞收缩力进行简化的高通量分析

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

医学

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

原发性患者特异性主动脉平滑肌细胞的分离和体外半定量实时收缩测量

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

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心外膜生长培养测定和