这项工作得到了中西部大学健康科学研究促进委员会资助，授予EEH和中西部大学研究与研究局局长助学金，授予KJL。附属实验室和附属皮肤科提供了额外的支持。我们感谢Sarah Potekhen，Jamie Barto，Stefani Fawks，Cody Jording，Stacie Schimke，Heather Kissel和Ali Zaidi的技术援助。

所有对人体组织样品的实验均经Western IRB（WIRB Protocol＃20142461）批准。用于这些程序的组织收集的单一标准是SCC的活检证实的诊断。原始IRB方案中没有概述排除标准，但未使用已知血源性传染病患者的样本。在收集皮肤组织，血液和唾液之前获得知情同意书。中西部机构审查委员会批准了在西西里大学（AZ＃807）的基于临床的生物样品样品的验证工作。 皮肤肿瘤组织加工注意:这是由莫氏组织技术专家执行的。 <ol><li> 新鲜鳞状细胞癌（SCC）组织RNA和外植体培养的加工 <ol><li>组织被Mohs外科医生切除后，收获10至50毫克（≥10毫米3） </sup&gt;）组织样品从松弛期间的组织顶端侧的中心（这是操纵组织以允许其平放在载玻片上的过程）。 注意:仅当肿瘤足够大以允许在组织学评估之前移除10-50 mg大小的样品（≥10mm 2 ），这是Mohs手术的一部分，才有可能。如果肿瘤不够大以至少要移除2mm 3 ，则不要进行RNA的处理（跳过步骤5）。 </li><li>使用镊子将活组织转移到含有培养基（DMEM:Ham&#39;s F-12,25mM HEPES，100IU / mL的青霉素和100μg/ mL链霉素的1:1混合物）的标记的1.5mL管中，并确保组织完全浸没在培养基中。将此管存放在4°C，然后再进行加工。 </li><li>使用组织进行RNA分离和表达分析（见步骤5）和/或建立在24小时内进行外植体培养（见步骤6）。 </li><li>将从患者中取出的肿瘤样品的剩余部分（步骤1.1.1）放置在低温恒温器组织支架（或"卡盘"）上。将组织嵌入最佳切割温度（OCT）化合物中，冷冻保持器内的组织，以允许完成莫氏组织学和制备用于实验分析的另外的组织学载玻片。 </li></ol></li><li> 组织学幻灯片的制备 <ol><li>使用低温恒温器切割7μm厚的组织切片，并为每个肿瘤样品创建两个（或更多）个载玻片。确保每个幻灯片最多包含完整肿瘤的三个部分。 注意:两个幻灯片中的一个将被Mohs组织技术学家用H＆E染色，另外的幻灯片可以用IF，IHC或FISH分析进行处理。 </li><li>将载玻片放入丙酮中1-2s以固定组织。将这些幻灯片放在一边干燥。不要H＆E染色或盖玻片以用于将来的免疫荧光（IF），免疫组织化学（IHC）或荧光原位杂交（FISH）分析，这取决于实验方案。 </li></ol></li><li> 后Mohs组织的处理 <ol><li>一旦Mohs处理完成（步骤1.1-1.2），在低温恒温室中工作，并从低温包埋介质中撬起样品并将组织置于标记的低温管中。 </li><li>在组织解冻之前，将标记的小瓶置于液氮中。如果将样品用于蛋白质和/或DNA分析，则可将其转移到液氮或-80℃冰箱中，以便将来储存以用于将来的蛋白质表达和DNA突变分析（参见步骤4-5） 。 注意:如果从Mohs后期样品中需要完整的RNA，则必须在将样品从冷冻包埋介质中取出时保持冷冻。如果不行使极端的照顾此步骤后Mohs样品应保留用于蛋白质和/或DNA分析。 </li></ol></li><li> 新鲜ANT的加工 <ol><li>在Mohs关闭时，从Mohs外科医生那里获得ANT。 </li><li>从正常组织的顶端取出从SCC样品（见上文）收获的等量或更大量的ANT，并将其转移到含有培养基（1:1混合DMEM:Ham&#39;s F- 12，补充有25mM HEPES，100IU / mL青霉素和100μg/ mL链霉素）。 </li><li>将组织完全浸入同一培养基中并在4℃下储存，直到后续处理（见步骤4-6）。 </li></ol></li></ol>血液，唾液和颊拭子加工<ol><li>通过静脉穿刺在EDTA收集管中获得约10 mL的血液。 </li><li> 将血液从收集管转移到无菌的15或50 mL离心管中。将大约200μL去除在1.5 mL低温管中，避免气泡;将这种全血样品储存在-80°C，如有必要可用于将来的基因分型和组织匹配。 <ol><li>将剩余血液以1,000xg旋转30分钟，并按照标准程序收集血浆级分。 </li><li>在1.5 mL低温管中将等离子体等分为1 mL增量，并储存于-80°C，以备将来进行液体活检分析。 </li></ol></li><li>使用无菌拭子获得颊拭子样品，并将拭子在室温下储存在无菌管中。 </li><li>如果需要，获得唾液样品，并在室温下储存在无菌管中。 注意:如有必要，这些样品可用于将来的基因分型和组织匹配。 </li></ol> 3.记录样品<ol><li>将患者样本清单中的信息转移到生物参考数据库。包括患者信息和诊断nosis从病人图。 </li><li>使用每个样品的条形码打印标签，并将数据记录在数据库中，以将每个收集的样本与患者数据相链接。 </li><li>在将样品转移到研究实验室之前，记录未确定样品在生物数据库中的分布情况。 </li></ol>蛋白质分离和蛋白质印迹分析<ol><li>将每个组织样品（SCC和/或ANT）放入含有每毫升组织含有蛋白酶抑制剂的500μL细胞裂解缓冲液的小瓶中以裂解组织。 注意:我们使用由50mM Tris pH 8.0,150mM NaCl，0.5％脱氧胆酸钠，1.0％NP-40和0.1％SDS组成的放射免疫沉淀测定（RIPA）缓冲液。可以使用其它细胞裂解缓冲液。 </li><li>使用组织匀浆器在4℃以20秒间隔使组织匀化5分钟。将样品在21,000 xg和4℃下旋转20分钟，并将含有蛋白质的上清液收集到新鲜的tu中是。使用相同的条件重新旋转上清液，并将最终的上清液收集到新鲜管中。 </li><li>使用4-20％SDS-聚丙烯酰胺梯度凝胶从每个样品中电泳30-50μg蛋白质。如果需要，用考马斯蓝染色凝胶以显现蛋白条带。 </li><li>如果需要蛋白质印迹分析表达的蛋白质，按照标准方案将蛋白质从凝胶转移到聚偏二氟乙烯（PVDF）膜。转移后，如果需要，Ponceau染色PVDF膜以显现蛋白条带。 </li><li>阻止PVDF膜和探针以按照标准方案检测目标蛋白质。按照制造商推荐的蛋白质印迹分析的浓度，使用对所关注的蛋白质特异性的一级抗体以及对于一级抗体特异的相应的二级抗体。 </li><li>使用化学发光底物暴露探测膜，并使用图像对膜进行成像ng系统。 </li></ol> RNA分离和定量聚合酶链反应（qPCR） <ol><li>立即将新鲜的组织样本（SCC和/或ANT）放入含有RNA稳定溶液的1.5mL管中，并将Mohs组织样品放入含有冷冻组织转换溶液的1.5mL管中，按照制造商的建议。确保组织完全浸没。将这些样品储存在-80°C直到使用。 </li><li>从-80°C取出RNA样品，并在冰上解冻。将组织转移到每50-100mg组织中含有1mL RNA提取缓冲液（苯酚/硫氰酸胍）的1.5mL管中。 </li><li>使用组织匀浆器裂开组织。进行六个循环的均质化，然后进行一段时间的样品冷却;每个循环由4℃均匀化和40s冷却期20s组成。均化后，在室温下孵育样品5分钟。 </li><li>加0.2mL氯仿/ 1mL RNA提取缓冲液，用手剧烈摇动管15秒。在室温下孵育2-3分钟。 </li><li>在12,000 xg和4℃下旋转样品15分钟。取出样品的水相并将其放入新的1.5 mL管中。 </li><li>每1mL步骤5.2中使用的RNA提取缓冲液加入0.5mL 100％异丙醇至水相，并在室温下孵育10分钟。 注意:如果RNA产量预计为低（ 即起始组织体积小于10毫克），样品可能会在-80℃下沉淀过夜。另外，在异丙醇沉淀步骤期间，可以加入每500μLRNA提取缓冲液中5μg无RNA酶的糖原，以提高RNA产量。 </li><li>在4℃下以12,000xg旋转样品10分钟。取出上清液，用1 mL 75％乙醇洗涤沉淀。 </li><li>短暂旋转样品，然后以7,500 xg和4°旋转C 5分钟弃去洗涤物并将其干燥5-10分钟。 </li><li> 将RNA沉淀重悬于20μL无RNase的水中。 <ol><li>如果需要，按照制造商的建议，使用RNA清洁试剂盒纯化每个RNA样品。 注意:如果执行此步骤，小RNA将从样品中丢失。 </li></ol></li><li>使用1.2％甲醛-MPS琼脂糖凝胶电泳1μg样品以分析RNA的质量。 注意:作为替代方案，使用生物分析仪分析样品以获得RIN编号13 。 </li><li>使用反转录方案将RNA逆转录为cDNA，以便对感兴趣的靶mRNA（或miRNA）进行qPCR分析。 </li></ol> 6.组织外植体培养<ol><li>在将组织浸入70％乙醇中之前，将培养前的组织进行灭菌。 </li><li>将组织放在载玻片或碟上，加入足够的培养基（DMEM:Ham＆＃39; s F-12，补充有10％FBS，25mM HEPES，100IU / mL青霉素和100μg/ mL链霉素）以覆盖组织（以防止组织干燥）。使用解剖刀刀片小心地将组织切成碎片（&lt;1 mm 3 ）。 </li><li>将组织碎片转移到35mm组织培养皿中，并在每个片段的顶部加入约20μL胎牛血清（FBS）。 </li><li>将培养皿放在培养罩中20-30分钟，或直到几乎干燥。 </li><li>向每个培养皿中加入1mL培养基（1:1混合DMEM:Ham&#39;s F-12，补充有10％FBS，25mM HEPES，100IU / mL青霉素和100μg/ mL链霉素），并在37℃下孵育C在5％CO 2中 。如果需要，进行亚文化或亚克隆培养。 </li></ol>

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作者没有什么可以披露的。

据笔者了解，该协议是首例以成本效益和速度快的方式重点关注皮肤组织样本的临床采购。经历莫氏显微外科手术的患者通常在特定的时间段内安排，收集限于这些时期。样本收集涉及病人护理中涉及的医疗助理，处理肿瘤样本的莫氏组织学技术人员以及处理样品进入和血液处理的指定实验室工作人员的努力。 收集队的三名成员（ 即医疗助理，莫斯组织技术员和实验室技术人员）之间的认真协调，我们发现现有的医务人员可以完成与收集3-5个组织样本相关的额外任务每天不会明显延误诊所的行动。获得患者同意<html>并且在Mohs显微外科手术涉及的正常等待期间完成收集颊拭子和血液。除了关于如何处理样品的决策之外，莫氏组​​织工程师负责的任务在患者样品的莫氏加工结束时完成，通常将每日计划增加约30分钟。通常，诊所工作人员中的一名成员负责对血样进行标签和处理，并执行将样品转入和进入数据库。根据处理的样本数量，这些职责可能需要相当长的时间远离现有职责;因此，只有在有足够的实验室人员配备的情况下才能进行样品采集。具体来说，如果在一天内收集了超过5个样本，或者从患者收集了几个血液样本，则需要从实验室技术人员进行数小时的额外努力。 在收集日，医疗助理（MA）获得患者捐赠组织的同意书，随后获得口腔拭子（如有必要）唾液拭子（唾液），并由实验室人员收集血液进行处理，不再有收集这些样本后患者参与，所以从病人的角度来看，临床访问的其余部分没有改变（尽管在任何随访期间要求他们捐献血液），目前我们可以估计70％的患者同意捐赠组织，约30％的患者也捐献血液，30％的患者参与减少，目前只有经活检证实的癌症诊断的患者才包括在生物制剂中。已知血源性传染病患者被排除。 实验室工作人员负责与收集组的其他两名成员进行协调，并记录收集样本（表1）。之后将初次样本收集，完成的同意书，血液和颊拭子转移到实验室工作人员，并记录捐赠者的姓名，出生日期和其他相关信息，如样本类型。这样可以在收集过程中记录每个患者收集的样本的数量和类型。总结SCC组织（ 图6 ）和ANT（图7）的处理流程图是收集程序中新员工培训的有用工具。 <img alt="图6" src="/files/ftp_upload/55583/55583fig6.jpg" /> 图6: 描绘SCC样品处理的流程图。肿瘤切除后，评估样本的大小。如果样品≤2mm3，则整个样品用于Mohs处理，作为清洁边缘的评估（标记）完全消除肿瘤）是重中之重。在Mohs加工之后，将任何剩余的组织在液氮中冷冻并转移到生物支架中。如果样品足够大，使得可以除去组织的&gt; 2mm 3部分，则将该部分收集并放置在含有细胞培养基的管中，而剩余部分用于Mohs处理并如上储存。将除去细胞培养基的样品部分进一步分段，除去约0.5mm 3片段，并用于建立细胞培养物系;将剩余的组织片段转移到生物支架用于下游蛋白质和基因表达分析。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55583/55583fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> er.within页= "1"&gt; <img alt="图7" src="/files/ftp_upload/55583/55583fig7.jpg" /> 图7: 描绘ANT样品处理的流程图。在Mohs闭合时，由Mohs外科医生去除并收集相邻正常组织（ANT）的一部分。如果样品尺寸≤2mm3，则将整个样品置于低温管中并在转移至生物支架之前在液氮中冷冻。如果样品&gt; 2mm 3 ，则将整个样品首先放入含有细胞培养基的管中。然后将样品进一步切片至〜0.5mm 3片段，用于建立细胞培养物系。将剩余的组织切片转移到生物支架中以储存用于下游蛋白质和基因表达分析。55583 / 55583fig7large.jpg"target ="_ blank"&gt; 请点击此处查看此图的较大版本。 该协议旨在最小化生物反应器中样品之间的变化。临床工作人员与诊所和所有实验室人员之间的协调和有效沟通对于最小化变异性并最大限度地从组织中获得高质量的DNA，RNA和蛋白质至关重要。虽然组织内的DNA和蛋白质是健壮的，但如果需要RNA，则在收集，运输和加工过程中保持组织冷和适当的培养基至关重要，以保持RNA的完整性。准确的记录记录对于将患者样本与其病史和诊断联系起来也是至关重要的，因为当分析来自下游应用的结果时通常需要这些信息。 随着对个性化医学和生物学的兴趣日增标记研究，最近已经建立了许多生物靶。这些通常与大型学术中心的医院诊所相关联，并且在设置和操作2,8,19中涉及大量成本。作为日常实践的一部分产生的作为皮肤癌样品的基于临床的生物制剂的开发具有优于其他生物栓剂的几个关键优点。首先，通过单个收集和采样处理站点，最小化可能危及下游应用程序的样品处理变化。第二，由于这些捐赠样本是从稳定的患者人群中抽取出来的，因此获得未确定的医疗记录和患者结果数据是无与伦比的。这些数据可以在样品收集时或在随访中被匿名化并从患者图表转移到生物参考数据库。第三，用忠实的病人基地，基于inic的生物原子能够从相同的患者收集多个样品，在其他设置中不经常获得的样品组。该方案的局限性之一是对通过莫氏显微手术获得的皮肤样本进行了优化，并且可能需要对其他类型的组织或临床程序进行修改。 总体来说，生物制剂的主要目标之一是提供来自同一捐赠者的广泛样品，使研究人员能够提出广泛的问题，特别是液体活组织检查和癌症标记物的开发。具体来说，将肿瘤组织学和生物标志物在mRNA和蛋白质水平的表达与血浆样品相关联的能力以及参考患者结果数据的能力创造出独特强大的数据集。我们希望以临床为基础的生物原理模型可用于帮助减轻生物医学研究中患者样本的不足。

癌症和生物标志物研究依赖于提供优质的人体组织样本，有限的供应阻碍了研究1,2 。许多皮肤病学研究受到供应不足，质量变化以及与使用人体组织有关的成本的限制。建立大型专用生物库的成本估计约为200万美元3 ，这些成本将人体组织的使用放在许多研究人员的触角之外。此外，生成和存储研究样本的过程有可能影响临床操作，并延迟患者护理，如果不慎重执行。已经建立了一个具有成本效益，基于诊所的生物反应器，其重点是按照推荐的最佳实践和样品验证4,5,6 。 <pclass ="jove_content"&gt;该协议已经在皮肤科诊所中开发，该诊所执行大量的Mohs显微手术以去除鳞状细胞癌（SCC），基底细胞癌（BCC）和黑素瘤皮肤癌。志愿者捐助者可以从这个病人群中招募。重要的是在收集部位建立生物反应器，以迅速从同意的患者中获取组织和血液，而不会延迟治疗。收集相同诊所的样品可最大限度地减少采集技术的变化，并最大限度地减少样品质量的变化，这对于下游应用7,8可能是有问题的。 莫氏显微手术技术的目的是确保所有的癌组织被去除，同时保留尽可能多的健康组织。该过程涉及逐渐去除肿瘤组织的薄层。每个连续的层是他的通过皮肤科医师进行病理学检查（在对肿瘤组织进行冷冻切片并进行H＆E染色后），以确定是否所有的癌组织都被去除。当患者留在办公室时，执行随后的组织层的切除和检查。该技术被认为是SCC 9的最佳治疗方案。此时，伤口闭合，为了改善愈合和美容外观，常常切除相邻的正常组织（ANT）。因此，这种去除肿瘤的外科手术理想地适用于收集组织学表征的组织用于将来的研究。 用于获得肿瘤组织，相邻正常组织，唾液和血液样本的采购程序被设计为对正常人员职责的影响最小（ 图1 ）。医疗助理在准备患者手术时进行抽血。在完成莫氏程序后，<html>哦，组织技术人员准备标本的附加组织学载玻片并将组织转移到生物支架。与建立生物反应器相关的成本包括购买冷冻保存冷冻机，创建适度的临床实验室空间以及开发库存跟踪计划。 <img alt="图1" src="/files/ftp_upload/55583/55583fig1.jpg" /> 图1:样本收集和负责人员的顺序。病人登记并取得病人同意后，医疗助理将收集颊拭子并进行抽血。然后皮肤科医师和医疗助理人员切除肿瘤并关闭伤口，在此期间分别收集SCC和ANT标本。专门的实验室技术人员处理血液和切片SCC和ANT标本，用于组织培养，保存和进入生物支架。ftp_upload / 55583 / 55583fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 收集的样品的多样性可实现多种实验方法（ 图2 ）。从患者收集的样品是颊拭子（如果需要，也可以收集唾液），全血和切除的组织。颊拭子和来自全血的样品在不加工的情况下被保存用于基因分型和组织匹配。将全血分离成白细胞（WBC）和血浆级分用于未来分析。在Mohs加工之后，将冷冻的肿瘤直接置于液氮中并转移至-80℃的冷冻器中。使用先前技术10,11的修饰培养新鲜可行的肿瘤组织和ANT样品，然后冷冻保存。在收集期间，每个样本类型的数量在进入之前记录在电子表格上<html>库存跟踪程序便于准确处理（ 表1 ）。 <img alt="图2" src="/files/ftp_upload/55583/55583fig2.jpg" /> 图2:基于临床的生物样品采集和处理的概述。从患者收集颊拭子和血液样品，并存储用于下游基因分型和组织匹配。全血进一步处理白细胞（WBC）分离和CTC分析，以及血浆采集和液体活检分析。在Mohs手术期间切除的组织进行组织学处理以用于诊断目的，之后组织学载玻片可用于实验用于进一步的免疫组织化学分析。假设切除的组织样本足够大，一部分新鲜组织被去除并切片以进行蛋白质和RNA分离，并建立培养的细胞系。55583 / 55583fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>收藏日期: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>病人1 </td><td>病人2 </td><td>病人3 </td><td>病人4 </td><td>病人5 </td></tr><tr><td>姓名和出生日期</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>眼睛的颜色</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>样品类型</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>样品位置</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>唾液</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> w ^洞血</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>等离子体</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>肿瘤生存</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>组织正常生存</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>肿瘤莫氏液氮</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>组织正常液氮</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>幻灯片</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表1:记录样品收集的清单。收集的每个样本跟踪和记录的数据包括患者姓名，出生日期和眼睛颜色（皮肤分类）以及位置取样。唾液样本的数量，采血量以及收集的活的和保存的组织标本的数量也被记录作为后续用途分配的参考。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55583/Table1.xlsx">请点击这里下载此文件。</a> 为了验证样品收集程序，已经在下游应用中测试了每种样品类型。使用先前技术的修改12 ，肿瘤和ANT已经成功地用于蛋白质和RNA分离，并且可以潜在地用于DNA分离。已经通过显微镜评估从组织切片建立的活的外植体，而存储的组织学载玻片已被用于免疫组织化学和免疫荧光。 通过遵循这里描述的方案，可以将该模型扩展到其他皮肤科诊所，其他肿瘤类型（如黑素瘤），以及其他外科专业和实践，为人类癌症的多方面研究提供人体组织样本。对于其他实践，可能需要对该协议进行轻微的修改，但原则上该协议适用于常规地丢弃患者治疗过程中收集的患者样本的任何外科手术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K3EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>02-685-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Double Edge Blades</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Medium Point General Purpose Forceps</td>
			<td>ThermoFisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza Walkersville KGM Keratinocyte Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9791321</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Tissue TEK OCT Compound</td>
			<td>ThermoFisher Scientific</td>
			<td>50-363-579</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1950 Cryostat</td>
			<td>Leica Biosystems</td>
			<td>14047743909</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Rapid-Chrome H&#38;E Frozen Section Staining Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>99-900-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Long-Term Storage Cryogenic Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>03-337-7X</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Boca Scientific BuccalT-Swab&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9679349</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Signaling Technology 10x RIPA Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>50-195-822</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>PI78443</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>05-401-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Morter Cordless Homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>12-141-3</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Free Pellet Pestle</td>
			<td>ThermoFisher Scientific</td>
			<td>121-141-364</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma SteriCycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>13-998-089</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>SH30071.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Advanced DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12-634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco 1M Hepes</td>
			<td>ThermoFisher Scientific</td>
			<td>15-630-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell Culture 35 mm with Vent</td>
			<td>ThermoFisher Scientific</td>
			<td>1256591</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CFH monoclonal antibody clone OX-24</td>
			<td>Abnova</td>
			<td>MAB12583</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p40 (p63 delta) antibody</td>
			<td>Abnova</td>
			<td>ABX-144A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti MUC-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-7313</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Snail + Slug (phospho S246)</td>
			<td>Abcam</td>
			<td>Ab63568</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phallodin</td>
			<td>Molecular Probes&#160;</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Molecular Probes&#160;</td>
			<td>P36941</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinic-based biorepository

在过去几年中，皮肤癌（ 例如鳞状细胞癌，基底细胞癌和黑素瘤）的发病率一直在增加。预计对于生物医学研究研究的皮肤肿瘤样本将会有平行的需求。然而，由于建立生物制剂的成本和缺乏可用于获得不干扰临床操作的临床样品的方案，组织的可用性是有限的。建立了一项协议，收集和处理在莫氏手术之日对常规临床手术影响最小的皮肤肿瘤和相关血液和唾液样品。 Mohs组织学技术人员从经历第一层莫氏手术的患者收集并加工肿瘤样本，用于活检证实的皮肤恶性肿瘤。在手术闭合时收集相邻的正常组织。可能收集的其他样品是全血和颊拭子。通过使用通常丢弃的组织样本，生成生物反应器，其通过基于临床和实验室设置提供了几个关键优点。这些包括广泛收集的样品;获取未确定的患者记录，包括病理报告;而对于典型的捐助者，在后续访问期间可以获得额外的样本。

肿瘤和ANT已经成功地用于蛋白质分离，并通过Western印迹进行测试。 如图3所示 ，可以在收集并存储在我们基于临床的生物反应器中的患者样品中检测到皮肤鳞状细胞癌标志物（Muc1 14 ，FHL- 115和p40 16,17 ）。 <img alt="图3" src="/files/ftp_upload/55583/55583fig3.jpg" /> 图3:从ANT和SCC提取的蛋白质的分析。从ANT和SCC样品中分离总蛋白，Western blot检测SCC已知标记物的表达。示出了粘蛋白1（MUC1），因子H Like-1（FHL-1）和p40（图B）的代表性的蛋白质印迹。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55583/55583fig3large.jpg" target="_blank">恳求</a>请点击这里查看此图的较大版本。 使用上述技术分离的RNA已经通过凝胶电泳，qPCR和RNA完整性分析进行了测试。图4显示了两个患者样品，从中分离出优质完整的RNA。 RNA适用于所有下游应用。 <img alt="图4" src="/files/ftp_upload/55583/55583fig4.jpg" /> 图4:使用生物分析的RNA完整性分析。通过凝胶电泳评估RNA大小和完整性（图A）。基于RNA样品的电泳痕迹，包括核糖体RNA与降解产物的存在或不存在的比例（图B），为每个样品产生RNA完整性编号（RIN）。 "10"的RIN表示完整的RNA样品，没有任何降解产物，而"1"标记完全降解的sample。对于来自SCC和ANT患者组织的代表性RNA样品，将带的密度计曲线绘制为荧光单位（FU）对核苷酸（nt）（图C）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55583/55583fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 通过显微镜评估从组织切片建立的活的外植体（ 图5A ）。这些由肿瘤和ANT样品产生的外植体培养物是混合的角化细胞和成纤维细胞培养物。这些培养物可能被传代培养或用于将来的细胞系发育。存储的组织学载玻片已被用于免疫组织化学和免疫荧光。 如图5B所示 ，SCC样品显示SCC18中的上皮 - 间质转化标记物的阳性染色。 图5:SCC切片的外植体培养和IF染色。将鳞状细胞癌组织外植体（图A，Scale bar =50μm）和作为Mohs显微手术的一部分聚集的组织切片的相对图像用抗pSnail / Slug一抗（B组，Scale bar =25μm ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55583/55583fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 我们已经通过实验测定，在RNA分离（步骤5）或外植体培养（步骤6）之前，组织可以在4℃下储存24-48小时而不影响结果。如通过凝胶电泳或生物分析仪和RIN编号评估的RNA产量或完整性不受影响。此外，使用存储在4℃下的组织可以容易地获得外植体培养物的时间长短这在诊所和研究实验室之间转移样品时提供了更大的灵活性。由于RNA分离和外植体培养在研究实验室中进行，而不是在临床上进行，所以这种特征使得基于临床的生物反应体系比我们以前预期的更为可靠。

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

经Mohs显微手术后，皮肤肿瘤经常被丢弃。这里描述了一种方案，使临床支持人员有效地处理和存储下游实验室应用的皮肤肿瘤（ 如鳞状细胞癌，基底细胞癌和黑色素瘤）样品，而不会干扰临床操作。

建立以临床为基础的生物制剂

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

establishment-of-a-clinic-based-biorepository

Nanyang Technological University

Research

JoVE Journal

Medicine

6.4K 次观看.  Midwestern University. 经Mohs显微手术后，皮肤肿瘤经常被丢弃。这里描述了一种方案，使临床支持人员有效地处理和存储下游实验室应用的皮肤肿瘤（ 如鳞状细胞癌，基底细胞癌和黑色素瘤）样品，而不会干扰临床操作。 

视频: Establishment of a Clinic-based Biorepository

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

在兔子中使用完全的白眼切除术建立严重干眼模型

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

建立小鼠重症急性胰腺炎模型，将牛磺胆酸钠逆行注射到胆胰管中

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

猪静脉移植病模型的建立与评价

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

建立简单有效的术中甲状旁腺成像大鼠模型

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

改良动静脉瘘手术建立 RV 体积超负荷小鼠模型

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

作者聚焦:出生后右心室容量超负荷小鼠模型的建立和确认  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.