作者要感谢的转化研究单位的技术人员;玛丽Economou，何塞·加尔万，卡罗琳锤，多米尼克·穆勒，利利安Schöni和信息学团队在病理学研究所，伯尔尼大学。

注：这项研究已批准因泽尔医院，伯尔尼，瑞士（13年7月10日）的当地伦理委员会。组织中的肿瘤银行伯尔尼伯尔尼大学，研究所病理科获得。 1，规划与设计（咨询） <ol><li>想想研究要回答的问题。决定对组织类型被包括在该项目。确定组织结构是很重要的回答这个问题。 </li><li>确定最有用的纤芯直径和每名患者的核上进行项目的数目。决定基于生物标志物的量的下一代的组织微阵列（ngTMA）的拷贝数进行调查。 </li><li>考虑统计方面，如样本量分析的组织微阵列（TMA）的构造之后。判断患者纳入本研究的数量和建立控制组织为阵列。 </li><li>获取组织（或诊断），苏木精和曙红（H＆E），每个病人的幻灯片。简要回顾幻灯片，并标识包含项目相关信息和组织学的人。 注意：请代替H＆E幻灯片的蜡块新的章节，如有特殊染色或免疫组化幻灯片所需的幻灯片扫描和注释， </li></ol> 2，幻灯片扫描<ol><li>打开电脑和幻灯片扫描仪和开放的扫描软件。选择“自动模式”从预览屏幕亮场扫描。 </li><li>点击“扫描选项”调整幻灯片的质量参数。在这个时候，选择是否幻灯片直接扫描通过网络或本地驱动器（或外部驱动器）访问的数字化平台，并设置扫描和聚焦点的数量的种类。 </li><li>点击“服务器参数”扫描到“案例中心”。在该步骤中，加入更多的杂志，标明所有的幻灯片，并设置在案例中心所在的文件夹幻灯片应存放。每本杂志最多可容纳25幻灯片。 </li><li>检查质量/实际组织切片扫描清洁度。如果需要，清洁载玻片用70％的乙醇。 </li><li>负载高达25杂志与幻灯片标签指向内（ 图3A） </li><li>将杂志扫描仪。超过一本杂志，将它们放置在彼此顶部。 </li><li>开始扫描幻灯片点击绿色箭头（ 图3B）。当所有幻灯片扫描，卸载杂志和关闭程序，PC机和幻灯片扫描仪。 </li></ol> 3，数码片夹标注<ol><li>输入数字幻灯片管理中心在浏览器地址并下载免费的数字幻灯片浏览器软件。 </li><li>打开滑盖数码播放器软件，并选择包含数字切片（ 图4A）的文件夹。 </li><li>添加注释，重新排序和管理以数位升幻灯片文件夹或分配用户权限的同事或添加附件的文件夹。 </li><li>点击它会出现在数字幻灯片查看器所需的数字幻灯片。 </li><li>使用放大工具，评估幻灯片并找到了融入ngTMA感兴趣的领域。 </li><li>使用“TMA注释工具”，选择所需的芯的尺寸和注解的颜色。通过点击它（ 图4B）放置在数字幻灯片注释。 </li><li>移动注释到所需的组织结构，以便将其纳入ngTMA（ 图4C）。 </li><li>通过使用注释工具，选择芯的尺寸和所希望的注释的颜色再次重复注解的过程。重复幻灯片浏览和批注上的文件夹中的所有幻灯片。 </li><li>或者，将组织核心的PCR扩增到0.2毫升管，而不是融入TMA。使用不同的颜色来标识标注幻灯片这些景点进行进一步的分子分析。 </li><li>创建的所有情况的列表及其相应的标注和颜色。 </li></ol> 4，组织芯片构建<ol><li>获取相应的石蜡组织块的所有注解的数字切片，排序为组织microarraying所需的顺序块。 </li><li>确保组织块有至少4毫米的厚度。若否，组织reembedding是必要的。 </li><li>打开PC“ON”和自动化组织微阵列点样仪。打开组织微阵列软件，并提供一个名称项目</li><li>执行“工具更改”，然后选择所需的刀具直径的项目（ 即 ，0.6，1.0，1.5或2.0毫米）。 </li><li>最多可以装入12&#39;收件人&#39;TMA块到机器中。得到的ID到每个12个块的</li><li>为每个收件人块TMA布局。观察TMA设计的行，列和制止号Y布线以及铁心之间的距离。为每个收件人阻止新的布局，或重复使用相同的布局。 </li><li>广场上的收件人块布局光标采取的第一个核心。 </li><li>接着，装入最多60供体块进入组织微阵列（ 图5A）。发生在每一行A到F 10块给每个块捐赠者的ID。观察各捐助块的图像通过组织微阵列自动获取在电脑屏幕上。 </li><li>与第一块开始，点击“幻灯片”。观察使用数字幻灯片查看器存储在数字幻灯片管理中心（或外部驱动器上）的带注释数字幻灯片。查看该供体块的图像和所述数字滑动面由端。 </li><li>该块图像上选择的参考点叠加捐助块图像与数字幻灯片</li><li>点击“下一步”后，观察该块的大图的注释。 （ 图5B &lt;/ STRONG&gt;）。点击标注的顶部确认，然后点击“开始”。 注意：这提示组织微阵列来启动在所选择的出发点在接收者块钻0.6mm的孔的直径。然后在第二步骤中，使用冲压工具，仪器冲头孔在从所选择的供体块中的组织在准确的注释和确认区域。内核然后从供体到受体块传输。 </li><li>经过大约500核心，使用清洁二甲苯棉签钻和冲压工具。 </li><li>如果组织内核所需的（而不是拳成ngTMA），点击“PCR”的工具块，确认达到4注释并点击“开始”。这将冲头和内核转移到0.2 ml管。 </li><li>重复点4.9至4.11与第二供体块，依此类推。 </li><li>使用所有捐助块后，进入第二轮捐助块（61-120）进入组织芯片呃，继续步骤4.8至4.11。 </li><li>刷新供体和受体的块图像，同时该项目的进展，同时钻孔，冲孔发生（ 图5C）。 </li><li>执行“导出”后，该项目已完成。找到了供体和受体块标识的.xslx文件，公会内的所有拳以及财资市场公会的布局/设计的本土化，在导出的文件夹中。保存所有捐助块图像与所有叠加标注为导出的文件夹（ 图5D）的JPG图片。最后TMA块，然后准备。 </li></ol>

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在本文中，协议ngTMA概述。 ngTMA是一家新成立的概念，涉及到规划，设计，数字病理学和幻灯片注释组织microarraying以及自动组织microarraying 12。 相比传统的组织microarraying，ngTMA提供了许多优势。在第一步骤中，规划和设计阶段是关键的。重点是回答有针对性的研究问题。这应该考虑到组织问题（ 例如 ，有多少点我想要的区域，包括？），统计规划（ 例如 ，样本量？如何后来我分析的样本？）和后勤方面的考虑（ 例如 ，如何许多生物标志物，因此多少ngTMA副本？）。具体ngTMA是为特定需要做，无论是生物标志物的筛选和高通量或拟研究特定组织学方面上只有少数几个精心挑选CASES。 一，如果不是，常规组织microarraying的最重要的缺点是低精度，这在组织切片的标记制成。具体的组织结构，细胞或地区的研究，取得了几乎是不可能的。 ngTMA允许高精确度，因为注释是对数字切片直接放置。这使得研究者能够精确地选择的区域被冲切，包括特定的细胞。在这个例子中，相同的组织块内的各种区域被冲切出的排列，例如在肿瘤中心，入侵前和肿瘤/基质相互作用的区域通过的小肿瘤细胞簇，甚至单个细胞中存在突出。此准确性只能使用ngTMA实现。第三，因为载片扫描到一个基于网络的数字平台，滑动观看和注解可以通过计算机而不是用显微镜进行。组织排列软件提供了一个用户友好的可以实现具有高度的灵活性，因此不同的布局和ngTMA设计接口。由于TMA建筑用钻孔自动执行的，有需要的小的动手操纵和时间进行施工的量显著降低。在这里这个例子的时候了公会建设是24小时之间。使用常规的TMA方法，并估计每个小时15拳，这个项目大约需要304小时。 ngTMA可应用于研究蛋白质的表达，mRNA或DNA，以及它们的组合， 图9示出几个这样的应用中的潜在生物标志物。标准免疫组织化学可用于确定使用Ki-67的癌症的增殖指数。的组合的方法来调查对基因如HER2蛋白表达和DNA扩增可以使用。组织可以一起聚集在一个单一的ngTMA以降低成本，组织使用和其它资源第此外， 在对HER2和其他基因原位杂交显色的mRNA可以被执行以确定在大量使用的组织切片的最小数目的情况下单个mRNA转录物。免疫标记物，如CD8 +免疫组化，可以可视化在肿瘤微环境的上下文。双免疫组织化学也可以用于突出显示感兴趣的特定区域，例如在癌症的侵袭前的免疫细胞（标记为红色）和肿瘤细胞（标记为棕色）之间的相互作用。这种利益的区域已经不能用传统的组织microarraying抓获。 然而，该协议包含了一些限制。最重要的挑战是供体块和数字幻灯片之间的重叠。有几个因素可以影响这一步。首先，从块的最新的部分应被用于幻灯片扫描。在很多情况下，H＆E不是供体块，rathe的最后部R在免疫组化或其他污渍。在这种情况下，也有染色的载玻片，可以作为最后的污渍被扫描和注释的或新的H＆E应作为长。当被制成组织切片的组织可以在水浴这导致滑动和块的具有挑战性的匹配扩展护理也应考虑。其次，在目前的项目仅限于在单一时间处理12收件人TMA块。必须在较大的项目超过12的TMA分配到第二个项目的名称。第三，捐助块必须使用标准的模具和磁带，仪器不能自行调节，以各种尺寸进行。最后，捐助块必须超过最低高度（4毫米），以达到最佳的钻孔。在某些情况下，这需要组织reembedding。 数以百计的出版物在过去的几年中突出的公会作为一个宝贵的工具，生物标志物的研究。的TMA已被用于研究肺13，结肠7，冰川AST 14，前列腺癌15，16胰腺，膀胱癌17，和胃的癌症18，仅举几例。越来越多的作家已经联合使用的TMA与图像分析和显著进展目前正在朝这个方向19-21。然而，少数已公布的创新意念的TMA 22-24研究小组的旁边，稍不注意被赋予了优化TMA技术本身。自动组织microarrayers，如ATA-27由Estigen /彻办提供版面设计和方便的和自动化的组织冲孔。然而这表示ngTMA概念的仅有1方面。 ngTMA是一个实质性的改进，传统的组织microarraying技术。它包含在组织学和TMA设计专长与数字化病理学的灵活性和数字标注的速度和自动化TMA建筑物可靠性的精度。 ngTM的组合A和图像分析，蛋白质和分子生物标志物的评估将是一个强大的工具来进一步提高临床和转化研究，在未来的质量。

在过去的二十年中，组织微阵列（的TMA）已对生物标志物的调查研究的显着影响。的TMA基本上由重复传送的小组织核心，通常为0.6〜2.0mm的大小范围在直径制备的组织“档案”，从石蜡包埋的“供体”的块为一个单一的TMA“收件人”块（ 图1）1。用一个小的核心面积，约500种不同的组织点从几个或许多不同的患者可排列为一个TMA 2。 用于预后或预测的生物标志物的研究中使用的TMA的具有很多优点。请看例子，其中的蛋白质生物标志物免疫组化法对表达对450名患者进行评估。而不是执行在450患者玻片从块的相同数目切片450免疫组化染色，从每个样品中的小核可以被排列到一个单个T马块。即使多个内核，从每个个体患者服用，块的最小数目被生产。这具有大大减少成本和其他资源，以及减少组织消耗的相当大的效果。使用大量组织的另外这允许适当地供电的研究，以在相同实验条件下进行评价。 的TMA具有许多不同的应用。例如，它们可以被用来研究形态，蛋白质表达，RNA表达和染色后用不同的染料DNA畸变，或免疫组织化学或发色，甚至荧光原位杂交3-7之后。最近的研究还利用组织芯片检测的染色方法和内部实验室间的差异，建立特异性抗体或特定基因突变的敏感度，并确定蛋白表达在国际合作的观察者间的重复性<suP&gt; 8-11。 传统的TMA使用来自患者的组织建设是一个长期的多步骤的过程（ 图2）。它从一个寻找可能的适当情况下并选择从档案诊断幻灯片在病理学研究所，或其他机构，从那里他们被检索。病理学家评估每个个案每张幻灯片，并选择最有代表性的幻灯片研究的目的。接着，对感兴趣区域直接使用显微镜下一支笔标记。这往往是具有挑战性和不精确而导致仅在组织中的冲头，应采取由一个“估算”。接着，对应于这些标记的幻灯片的石蜡块中检索从归档。块和滑块之间的快速对比。使用半自动化或自制的组织阵列中，供体块被冲压出在感兴趣的估计区域，并转移到受体TMA块。组织芯片采用这种排列方法的施工劳动强度大，费时，不精确和不灵活。准备475斑点TMA 3份，估计需要约84个小时的工作。 规划设计（或咨询），数字病理结合专业知识，组织学和自动化TMA排列12：一种新的方法来组织芯片的构建近日由病理学研究所，伯尔尼大学，依靠三个组件介绍。总之，这个概念被称为新一代的组织微阵列（ngTMA）。下面，一个协议，用于ngTMA是基于对134例转移性大肠癌的一例进行说明。这里，原发肿瘤以及淋巴结转移和肝转移要被排列成ngTMAs用于随后的生物标志物分析。此外，从每个病人的小组织芯被期望为未来的核酸提取。

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a next-generation tissue microarray (ngtma) protocol for biomarker studies

生物标志物的研究依赖于组织微阵列（TMA）。 TMA被由小组织核心重复传输从“供体”块变成了&#39;收件人&#39;块制作，然后用于各种生物标志物的应用。传统的TMA建设是劳动密集型的，不精确的和耗时的。在这里，使用新一代的组织微阵列的协议（ngTMA）概述。 ngTMA是基于TMA的规划和设计，数字病理学和自动组织microarraying。该协议使用134转移性结直肠癌患者的一个例子示出。组织学，统计和后勤方面考虑，如组织型，特定组织区域，并且以包括在TMA，组织斑点，样本大小，统计分析的TMA的份数，及许多细胞类型。组织学幻灯片每个病人进行扫描并上传到一个基于网络的数字化平台。在那里，他们被认为与安otated（标记），使用0.6-2.0毫米直径的工具，用不同的颜色来区分组织区域多次。供体块和12&#39;收件人&#39;块装入仪器。数字切片检索和匹配供体块的图像。注解区域的重复排列，自动执行产生一个ngTMA。在这个例子中，6 ngTMAs计划包含六个不同的组织类型/组织区域。两个副本ngTMAs的期望。三到四个滑动针对每个患者进行扫描; 3扫描运行是必要的，在一夜之间完成。所有的幻灯片都注明;不同的颜色来代表不同组织/区域，即肿瘤中心，侵袭前，肿瘤/基质，淋巴结转移，肝转移，与正常组织。 17注释/箱制成;时间标注为2-3分钟/箱。 12 ngTMAs生产含4,556点。排列时间为15-20小时。由于它的精度，灵活性和速度，ngTMA是一个功能强大工具，进一步提高组织芯片质量应用于临床和转化研究。

规划与设计这里使用的134例转移性结直肠癌，正在调查针对一系列特定的生物标记物的例子。不仅是一个ngTMA计划在这些患者中，但DNA的提取，随后突变分析为某些特定基因。 在ngTMA的规划和设计阶段，从以下几个方面均已确定。从每一位患者，6个不同组织的地区进行调查：肿瘤中心，肿瘤侵袭前，最密集的肿瘤领域崭露头角的（肿瘤/基质的相互作用），正常组织，淋巴结转移和肝转移。每个肿瘤组织区三个肿瘤点和两个正常组织点要包含到项目组（n = 17点每名患者）。因为按照计划，50多生物标志物放在这个病人材料研究，最终ngTMA的两个副本所需。由于淋巴结和distan的可能的小尺寸吨转移，0.6毫米的TMA建设小芯径为所有的组织选择。 此外，按照计划，每个区域单独排列，使6 ngTMAs含有不同的组织学领域将产生：一个ngTMA包含来自一）肿瘤中心组织，B）侵袭前，C）肿瘤芽领域，D）正常组织，E）淋巴结转移，且f）肝转移。 对于使用0.6mm的工具，TMA的设计，为0.4毫米拳之间一个舒适的距离将被选中。因为组织的斑点在长的行和列的评价通常是烦人的，两种不同的设计选择。对于每个组织区域3拳的肿瘤块中，创建一个包含六个部分的设计。这导致了402组织冲头的总数。对正常组织的冲头，每壳体2的冲头将被包括。布局装修432拳总的选择。 此外，对于每一种情况下，至多4个内核为0.6 MM会被额外冲出并放入0.2毫升管。这些组织核心也将被注明。 概括地说，6 ngTMAs不同组织会产生（5肿瘤数组x 3拳点¯x134例1的正常组织阵列×2冲点¯x134例）除了每名患者4芯的管中。综合来看，2814注解将会作出修改。 其次，从选择的患者相应的H＆E幻灯片检索从幻灯片存档。每个案例进行了简要由病理学家审查，并组织各类型的最具代表性的幻灯片被选中。 扫描每个组织切片的数字载玻片为未来注解制成。对于本研究，3-4组织切片均为每个情况下， 即原发性结直肠癌进行扫描，具有或不具有相邻的正常组织，淋巴结转移和肝转移以及可能的附加 ​​幻灯 ​​片CON泰宁正常粘膜。 多达10种不同的杂志，可以完全加载到幻灯片扫描仪;因此，250的幻灯片可以扫描在单次运行。时间对扫描和数字幻灯片大小随所述组织和所需的质量因子的尺寸。质量因子的范围从0到100这里，60的品质因数被选择的，因为它产生较小的扫描文件的大小没有扫描质量的任何显着损失。小活检可在30秒内进行扫描，而整个组织切片，如在本实施例中使用可能需要2至10分钟的那些。数字幻灯片大小也可以从2 MB到2 GB。使用60的品质因数，扫描600 MB和1 GB的生产之间。 与402和536的幻灯片，需要为这个项目总共3扫描运行。每次运行过夜进行。大约需要500 GB的存储空间。幻灯片直接扫描到一个名为案例研究中心的数字化平台（ngtma.unibe.pathcasecenter）。案例中心是可从与Web访问的任何地方通过输入指定的用户名和密码。 注释为注解，两个方面，应考虑：1）不同的组织区域应给​​予不同的注解颜色和2）的注释的数量应反映最终ngTMA所需的份数。 在这种情况下，3点每肿瘤区域的单拷贝已经被选择。对于2份，每个区域必须使用6个点进行注释。有用的是绿色为正常组织，蓝色为肿瘤中心，黄色为肿瘤侵犯前，红色为肿瘤的萌芽（肿瘤/基质）的相互作用，黑色为淋巴结转移，绿松石的远处转移，最后白色的区域是区域用于管。每个案件的注释，需要2-3分钟。 ngTMA建设这项研究需要6 ngTMA块吨Ø在2份建造，导致最后的12块。收件人的块，可以由样仪所支持的最大数目是12。每个肿瘤块将包含402组织的斑点和各正常组织块将包含268点，共计4556点在整个项目。 àTMA布局为每个收件人块中创建和保存项目（ 图6A，6B）。 60组织块可以被装载到组织微阵列平行;每个供体块的图像被拍摄。与第一供体块开始，相应的注释数字幻灯片从案例中心检索。滑动匹配被执行时，其可以1-3分钟之间取。注释确认和样仪的提示，开始打孔（ 图7）。核心冲压和传输时间大约为12秒。内核在此过程中的损失是最小的。在排列这个项目的总时间大约为24小时，其中包括时间SLI德/块匹配和仪器的重载。 ngTMA的一些例子示于图8A中 。芯用于随后的分子分析，也可以冲压出此时的（ 图8B）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig1highres.jpg" width="500" /> 图1：A）&#39;捐助&#39;块。从供体块核心是冲压出来，转移到二）收件人块，或组织微阵列（TMA）。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig2highres.jpg" width="500" /> 给病理学家F 图2。传统组织芯片的工作流程。一）组织学幻灯片从档案和 B检索）<html>或审查。 光盘 ）注解在幻灯片上显示的&#39;估计&#39;区域转移进行。 五）相应的组织块进行检索。 六）块和滑动进行比较的匹配，这可以G）有时是一个具有挑战性的任务。HJ ）组织microarraying再发生。K）的核心是不断转移和L）最后TMA构造。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig3highres.jpg" width="500" /> 图3：A）最多25幻灯片可以被放置在每个暗盒用于扫描。B）中的调整的品质因数和尺寸可以制成。扫描进展进行监控。 “SRC =”/文件/ ftp_upload / 51893 / 51893fig4highres.jpg“WIDTH =”600“/&gt; 图4。）扫描的幻灯片可以上传到基于Web的平台，案例中心。 二）数字幻灯片可以看到。财资市场公会注释工具允许用户选择所需的标注。 三）注释可直接放置到数字幻灯片的核心大小和颜色。 <a href="https://www.jove.com/files/ftp_upload/51893/51893fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig5highres.jpg" width="500" /> 图5 A）最多60块可以加载到组织微阵列，即每行10供体块和12个接收者块为好。B）中的每个供体块中的图像被拍摄时，以数位升滑动检索。块和滑动的匹配行为和说明用于块冲孔。℃）的排列的进行可以在仪器冲孔来监测。D）中进行冲压后，将各点的位置的布局中的一个出口，但也有相应的注解的每个供体块的图像被制成。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig6highres.jpg" width="500" /> 图6。这些TMA布局被用于排列在A）的肿瘤块和B）正常组织。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig7highres.jpg" width="500" /> 组织微阵列软件的图7。截图。在左边，12重cipient块可以被放置在仪器上。在底部，每个供体块的图像被拍摄。在该中心，财资市场公会的布局，发现了冲孔的进展以及与注解每个供体块的放大图像。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51893/51893fig8highres.jpg" width="500" /> 图8。）新创建ngTMA块的一些例子。 二）其他组织的拳可采取后续的分子分析。这些内核冲出，并放置于0.2ml试管。 <img alt="图9" fo:content-width="6in" src="/files/ftp_upload/51893/51893fig9highres.jpg" width="600" /> 图中ngTMA生物标志物的应用实例9，Ki67蛋白在结直肠癌 ）免疫组化染色。贺双R 2免疫组织化学/ 原位表示B）的蛋白（棕色低表达）和未放大的HER2基因（黑色）相对于着丝粒（红色）的杂交测定℃）高表达的蛋白（褐色）和HER2基因扩增（黑色）相对于着丝粒（红色）。D）的显色中的Her2的原位杂交显示出在乳腺癌ngTMA单一的mRNA转录。 E）免疫组化的CD8 +在大肠癌。 F）的双重免疫组化染色泛细胞角蛋白标记AE 1 / AE（棕色;肿瘤细胞）和CD68（红色;巨噬细胞）强调在大肠癌肿瘤微环境<a href="https://www.jove.com/files/ftp_upload/51893/51893fig9highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies at JoVE.com

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

该协议的目的是优化建设和组织芯片质量的生物标志物的研究。它包括规划设计，数字化病理学，虚拟幻灯片注释，并自动组织排列的各个方面。

生物标志物的研究下一代组织微阵列（ngTMA）协议

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

a-next-generation-tissue-microarray-ngtma-protocol-for-biomarker

Research

JoVE Journal

Medicine

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

23.8K 次观看.  University of Bern. 该协议的目的是优化建设和组织芯片质量的生物标志物的研究。它包括规划设计，数字化病理学，虚拟幻灯片注释，并自动组织排列的各个方面。 

视频: A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

特里尔社会压力诱发心理应激的测试协议

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

科研

医学

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

供体角膜内皮角膜移植术的组织准备

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

人体胰腺的收集协议

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

联体小鼠：一个详细的协议

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

在组织变性/再生和癌症的研究利用小鼠诱导端粒酶等位基因

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) may detect adverse events of interventions such as fluid overload at an early stage. The Simple Intensive Care Studies (SICS) is a research line that focuses on the prognostic and diagnostic value of combinations of clinical variables.
The SICS-I specifically focused on the use of clinical variables obtained within 24 h of acute admission for prediction of cardiac output (CO) and mortality. Its sequel, SICS-II, focuses on repeated evaluations during ICU admission. The first clinical examination by trained researchers is performed within 3 h after admission consisting of physical examination and educated guessing. The second clinical examination is performed within 24 h after admission and includes physical examination and educated guessing, biochemical analysis and CCUS assessments of heart, lungs, inferior vena cava (IVC) and kidney. This evaluation is repeated at days 3 and 5 after admission. CCUS images are validated by an independent expert, and all data is registered in an online secured database. Follow-up at 90 days includes registration of complications and survival status according to patient&#39;s medical charts and the municipal person registry. The primary focus of SICS-II is the association between venous congestion and organ dysfunction.
The purpose of publishing this protocol is to provide details on the structure and methods of this on-going prospective observational cohort study allowing answering multiple research questions. The design of the data collection of combined clinical examination and CCUS assessments in critically ill patients are explicated. The SICS-II is open for other centers to participate and is open for other research questions that can be answered with our data.

Structured protocols are necessary to provide answers on research questions in critically ill patients. The Simple Intensive Care Studies (SICS) provides an infrastructure for repeated measurements in critically ill patients including clinical examination, biochemical analysis and ultrasonography. SICS projects have specific focus but the structure is flexible to other investigations.

简单重症监护室内反复临床检查和重症监护室超声观察性研究方案

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

一种猪异位心脏移植方案，用于向心脏同种异体移植物提供治疗药物

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...