Name: in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse
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Description: Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse at JoVE.com

Dr. K. Yuan was supported by an American Heart Association Scientist Development Grant (15SDG25710448) and the Pulmonary Hypertension Association Proof of Concept Award (SPO121940). Dr. V. de Jesus Perez was supported by a career development award from the Robert Wood Johnson Foundation, an NIH K08 HL105884-01 award, a Pulmonary Hypertension Association Award, a Biomedical Research Award from the American Lung Association and a Translational Research and Applied Medicine award from Stanford University.

伦理学声明:涉及动物主题程序已在医学院斯坦福大学医学院被批准的机构动物护理和使用委员会。 注意:动物是在用3％的蒸发器异氟醚和O 2气体的3％的电源麻醉。眼睛上的药膏兽医使用可能有助于防止干燥时的麻醉下。 1.细胞的制备<ol><li>培养人类内皮细胞和周细胞的细胞培养100 MM平板用10毫升适当的媒体。更换10毫升新鲜培养基每2 - 1天，直至细胞达到80％汇合。 <ol><li>培养的内皮细胞（EC）的补充有公司完整内皮细胞培养基（ECM）提供5％牛胎儿血清（FBS），10％青霉素/链霉素和10％的内皮细胞生长补充。 </li><li>在补充有公司完整周细胞介质（PM）的培养周细胞供给2％FBS，10％青霉素/链霉素和10％周细胞生长添加剂。 </li&gt; </ol></li><li>准备细胞混合物<ol><li>计算实验组和对照组的所需数量的细胞。 注:实验组每个插件都包含一个万美元（1×10 6）人内皮细胞和二十万（2×10 5）周细胞。对于对照组，每组插头包含120万只的人类内皮细胞。阴性对照组仅具有基质凝胶。每个组应该包括至少四塞，这将需要两只小鼠作为插头可注入的小鼠的腰部两侧。三个插头将作为一式三份进行实验和第四可能的情况下，一个发生故障被使用。准备25％的额外细胞相匹配凝胶的额外容量。 </li><li>收集和培养板细胞计数。 <ol><li>要取下电池，取出媒体和室温1X磷酸盐缓冲液（PBS）洗一次。除去PBS，加入1ml 0.25％胰蛋白酶/ 2.21毫摩尔EDTA，并孵育1分钟。加入2洗涤细胞断板毫升培养基，并收集在一个15毫升管。 </li><li>算使用根据制造商的说明血球细胞。 </li><li>离心细胞的适当数量的成丸，在400×g离心5分钟。为实验组，离心既内皮和周下来连成一片沉淀。 </li></ol></li></ol></li></ol> 2.基质凝胶制备<ol><li>解冻完全基质凝胶在4℃下4小时或过夜，得到均匀的溶液。没有混合是必需的。 </li><li>在1.5ml管分装基质凝胶和储存在4℃。 </li><li>预寒意无菌1.5毫升管和28克1在4℃毫升胰岛素注射器。注意:保持基质胶冰在任何时候。 </li><li>混合与bFGF的冷基质凝胶在1的体积比:100（bFGF的终浓度= 0.5微克/毫升; bFGF的储备溶液= 50微克/毫升）通过上下吹打数次混合矩阵凝胶溶液后，在冰上孵育30 - 60分钟b安伏与细胞在步骤3.1中混合。 注:混合前解冻股票的bFGF。计算所需要的注射插头的数量。例如，每个插头需要200微升基质凝胶。因此，准备25％的额外凝胶。 </li></ol> 3.混合矩阵凝胶细胞<ol><li>对于每个组，重悬细胞沉淀（含有适当的细胞数）与含有bFGF的母质凝胶的计算量。轻轻混匀，避免起泡，直到小鼠注射准备在15ml试管离开冰。 </li></ol> 4.鼠标准备<ol><li>在感应腔麻醉在3％异氟醚5分钟加3％O 2 1 SCID小鼠。集中在一个实验组，或2小鼠，一次。 </li><li>从感应腔在加热垫腹侧移开鼠标，地点向下并迅速经由非再呼吸系整个仁济供给麻醉附加一个吻喷嘴到鼠标的脸ction过程。切换从感应腔室到非再呼吸系统中的气体流量和降低气体压力，以1.5％的异氟醚和1.5％ 的 O 2。 </li><li>从两侧后区（大约直径1厘米），用剃刀或脱毛霜清除头发。 </li><li>擦拭用70％酒精垫皮肤区域。 </li><li>与第二只老鼠4.4 - 返回鼠标感应室，并重复步骤4.2。 </li></ol> 5.矩阵注射凝胶<ol><li>通过附着到非再呼吸系统和加热垫腹侧放置向下准备一只小鼠注射。 </li><li>加载基质凝胶成预冷的28克1毫升胰岛素注射器一次一个鼠标。 <ol><li>负载两个插头（每头200微升加25％的额外）到注射器矩阵凝胶的完整的500微升的体积。 </li><li>确保将细胞装载到注射器，以防止在CE的3分钟内注入基质凝胶到小鼠从沉降到注射器的侧面并防止基质凝胶从注射器的内部固化LLS。 </li></ol></li><li>抬起背部皮肤定位皮下空间，然后注入200微升基质凝胶的缓慢而均匀地进入左肋背面后的皮下空间。 </li><li>取出注射针慢慢地，小心翼翼地防止基质凝胶漏出注射部位。撞了会形成在注射的部位。擦拭注射部位用酒精垫。 </li><li>重复注射步骤5.2 - 5.5对小鼠的背部的另一侧，然后离开鼠标在它的后面，持续1分钟，以允许在热垫的基质的凝胶的凝胶化。 </li><li>介绍了使用永久性标记后14天内，以确定磕碰都颠簸。经过一番头发长回来，它会更容易找到肿块。 </li></ol> 6.基质凝胶插件隔离:注射后14天<ol><li>通过将动物安乐死人道的老鼠s到&gt; 5分钟吸入二氧化碳颈椎脱位，以确保死亡。 </li><li>卸下鼠标的背部基质胶栓。 <ol><li>轻轻地取出周围注射部位其插头重复步骤4.3重新长出位于毛。标记线表示原始凸点的皮肤下的大小。 </li><li>切除插头分别安装在插头的顶侧和底侧，周围皮肤和肌肉层沿。 <ol><li>使用精细手术剪，使上即垂直于皮肤插头的明显边界的切口，并通过切向插头下方的肌肉。然后，小心地切绕沿标记的边界的插头的外周。 </li><li>拔出插头，保持它夹在皮肤和肌肉层之间。在小烧杯用1X PBS立即冲洗洗去多余的血液。该插件现在可以是固定的（下一步，6.3）。 </li></ol></li></ol></li><li>修复PLU克4％的多聚甲醛。 <ol><li>将整个插头，包括皮肤和肌肉层，在50ml管中，用25ml 4％的多聚甲醛在室温下24小时而不摆动。第二天，转移插头成25个1ml 70％乙醇的供石蜡包埋之前另外24小时。 注:处理多聚甲醛慎用。戴手套。 </li></ol></li></ol> 7.石蜡过程中的基质凝胶插件<ol><li>准备石蜡包埋插头。 <ol><li>定向插头， 如图1a中 ;放置插头在一个组织盒，使得插头的侧面朝上，皮肤和肌肉层都是横跨组织块的表面，将第一个切片中被切割可见。 </li></ol></li><li>石蜡根据标准石蜡包埋协议13嵌入插头。 </li><li>削减8 - 10微米厚的部分离组织块，并安装到显微镜载玻片雅鼎标准石蜡切片协议13。 </li><li>贮存于室温阴凉干燥处的幻灯片，直到准备染色。 </li></ol> 8. HE染色（H＆E）染色第13 <ol><li>通过100％二甲苯传递载玻片两次去paraffinize组织切片，100％的乙醇，95％乙醇，70％的乙醇，最后1×PBS中，每一个用于在室温下5分钟。留在1×PBS中滑动，直到下一个步骤。 </li><li>吸取100微升H＆E解决方案的上段和孵育1 - 5分钟，或直到有作为在放大10倍的直立光学显微镜下观察细胞细胞核的蓝色和粉红色之间的细胞质鲜明对比。小心不要让H＆E解决方案触及目标。 </li><li>洗涤H＆E溶液关闭的扣篮在一个大烧杯中的自来水的滑动的滑动件，然后将滑动在烧杯齿条和与2分钟自来水冲洗。 </li><li>莫五个步骤9.8安装的幻灯片。 </li></ol> 9.染色其他幻灯片人类毛细血管和周<ol><li>通过100％二甲苯传递载玻片两次去paraffinize组织切片，100％的乙醇，95％乙醇，70％的乙醇，最后1×PBS中，每一个用于在室温下5分钟。留在1×PBS中滑动，直到下一个步骤。 </li><li>煮抗原检索解决方案的部分。 <ol><li>在一个2升的烧杯中，准备在95℃的加热块上沸腾抗原使用1倍的柠檬酸缓冲液（pH7.0）中检索溶液。 </li><li>一旦沸腾，将载玻片在金属机架上，并在20分钟的煮沸抗原修复溶液淹没。 </li><li>小心地取出从仍然浸没幻灯片加热块的烧杯中，并让溶液慢慢恢复到室温约30分钟。 </li><li>放置在1×PBS中滑动，直到下一个步骤。 </li></ol></li><li>方框在封闭缓冲液（章节1×PBS中，5％山羊血清，00.3％的Triton X-100）。 <ol><li>周围画有PAP笔每一部分的边缘疏水圆。 </li><li>放置在用蒸馏水湿度托盘的幻灯片在托盘的底部。 </li><li>吸移管将100μl封闭缓冲液到确保所有的组织的切片被流体覆盖（可能需要超过100微升）。 </li><li>孵育部分为1小时封闭液。 </li></ol></li><li>孵育初级抗体部分。 <ol><li>备具有两个初级抗体的一个解决方案探测CD31（内皮细胞），并在稀释缓冲液中稀释平滑肌肌动蛋白（周细胞）的每个部分（1×PBS，1％BSA，0.3％的Triton X-100）。 注意:在此，抗CD31浓度为1:50和抗平滑肌肌动蛋白浓度为1:300。 </li><li>孵育切片在4℃下在含有蒸馏水的湿度盘100微升第一抗体过夜。 </li></ol></li><li>洗幻灯片三次1X PBST（1X PBS + 0.1％TWEEN20），每次5分钟洗涤。 </li><li>孵育二级抗体部分。 <ol><li>准备用荧光团缀合的山羊抗兔第二抗体的溶液以识别在稀释缓冲液中稀释的抗CD31兔抗体。 <ol><li>注:平滑肌肌动蛋白的第一抗体已结合有CY3荧光团，所以不需要第二抗体染色。山羊抗兔次级抗体浓度为1:250。 </li></ol></li><li>孵育切片1小时，在含蒸馏水的湿度盘100微升次级抗体。 </li></ol></li><li>洗玻片三次在1×PBST，每次5分钟洗涤。 </li><li>装入幻灯片。 <ol><li>吸取2 - 3滴（〜40 - 60微升）与DAPI核染色防褪色安装解决方案，在每个部分。 </li><li>轻轻地放在一个没有。 1.5盖玻片在部分和安装的解决方案，从而允许流体在部分展开和向的边缘盖玻片。轻轻用手指压出气泡可能位于在截面。 </li><li>让幻灯片处理之前干燥至少1小时。 </li></ol></li></ol> 10.量化毛细血管密度和结构14 <ol><li>算在H＆E染色的切片的毛细血管的数目，表示为＃/毫米2。 <ol><li>获取下放大40倍一个堂堂正正的光学显微镜四个不同领域的照片。 注:毛细管被标识为管充满红血球。 </li><li>可替代地，代替手工计数毛细管，使用图像分析软件（ 例如，Wimasis图像分析）来量化各种血管生成的参数，如容器数，平均容器的长度和直径，和分支点。 </li></ol></li><li>免疫荧光染色切片分析毛细结构。 <ol><li>获得四个不同领域的图片在一个倒置荧光显微镜与FITC和TxRed过滤立方体。 <ol><li>在488纳米波长使用FITC立方体图像内皮通过激励和激发在594纳米波长使用TxRed立方体图像周细胞。 <style type="text/css">p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Arial}
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No competing financial interests enclosed.

基质凝胶塞法已被证明是评估在血管发生， 在体内血管生成和抗血管生成的化合物基因调节，并以补充在体外试验方便和有效的方法。这里，我们详细描述人类血管生成的新颖基质凝胶塞测定该调查期间血管形成的内皮细胞和周细胞之间的相互作用。 还有在这个协议的几个新颖的关键步骤。在低通道（通道1 - 4）细胞是优选的，因为在14天的实验设置中年轻细胞会更可行。使用内皮细胞的数目是最小的一个万元和EC中以周细胞的比例为5:1;然而，增加周细胞的细胞数量将加强毛细血管的覆盖面。为实验组单独的内皮细胞，注射的细胞的总数应内皮细胞和周细胞的总和，这是120万，为此即，每插头细胞总数是一致的。它来计算细胞数量和基质凝胶体积的适当的量是重要的。基本公式为1.2万个细胞在200μl基质胶。此外，在注射前，保持枪头，注射器，基质凝胶，并将细胞沉淀在冰上在任何时候。因为基质凝胶是粘性的，并且为了避免气泡的细胞悬浮液中成形，切成约1厘米的关1毫升枪头的尖端，以允许更好的流动。直到老鼠是准备注入请勿混用细胞基质胶。每个插头注射应少于一分钟。一个鼠标可以携带两个插头，一个在其背面的两侧。当提取插头一定要保持夹在肌肉和皮肤层之间的电源插头，然后基质胶栓会得到很好的隔离后收集。 基质凝胶塞测定法的缺点是，它是耗时的，昂贵的，并且涉及乏味和微妙的步骤如injec重刑和插件隔离。如果这些步骤发生故障，结果会被毁坏并且过程将必须用新的鼠和材料再次重复。 但是，数据是可重复的，并在实验的设计方面提供了灵活性。例如，生长因子或抑制剂可以在血管发育的不同阶段，可用于药物验证施用到插头。此外，其它类型的细胞，如生长停滞的细胞，转染的细胞系或肿瘤细胞系，在基质凝胶塞的掺入是血管生成过程中操纵内皮细胞表型的另一种可能性。我们的协议利用这种灵活性，并介绍了人源性周细胞内皮细胞一起，允许在体内形成功能齐全，覆盖周细胞，混合型血管。我们的协议还介绍了如何从分析这些插头组织切片关键血管生成端点，包括管数和管长，与存在这两种类型的细胞。

血管生成是由该新的血管从预先存在的血管网1在许多领域从正常发展到疾病形成和是正在进行的研究的焦点的过程。这种动态过程涉及的增殖和血管内皮细胞（ECS）和周构建血管管的朝向需要的氧气和营养输送2点定向招募的迁移。为了研究这个过程需要一个同样动态测定中，最重要的一个可以概括的管道形成三维性质。 体外 3D基质测定已经开发了满足这种需要，并已运作良好，使研究人员以限定在空间和时间上离散的步骤，其中的血管生成发生3，4，5，6。然而，这些体外 3D基质模型限于研究非灌注容器的ð因此缺乏相关的血管生成过程中的关键组件（ 例如，循环增长和抑制因子，非自然的紧张/势力横跨血管床），并不能模拟存在于活组织的复杂环境。为了解决此限制，几个体内血管生成测定法已开发7，包括基质凝胶塞测定法，其将是我们的报告8,9的焦点。 基质胶栓法是一个行之有效的体内血管生成实验，呼吁研究人员，因为它提供了一个强大的平台，以测试在不同的血管细胞和物质的作用。基质凝胶是由在37℃下固化成凝胶样物质的Engelbreth-Holm的-群（EHS）小鼠肉瘤细胞系分泌的市售基底膜溶液。基质凝胶可与细胞和/或物质，如生长因子，和麟蹄混合皮下反恐执行局到鼠标。主机内皮细胞会侵入插头超过14天，形成血管网，并成为与宿主的血液灌注。迄今为止，基质凝胶塞测定已专注于内皮细胞行为的血管生成过程中的研究，但是，据我们所知的任何努力尚未作出判断该测定是否可用于共培养的内皮细胞和周细胞研究这两种细胞类型的血管生成过程中如何相互作用。具体地，理解的EC和周之间的关系是用于研究疾病，其中血管损失是病理性的，包括微血管缺血和周围血管疾病10，11，12有价值。 在这里，我们描述了引入了源于人的周细胞与人类内皮和成纤维细胞生长因子（bFGF）沿着矩阵凝胶混合物的协议。该混合物然后可以皮下注射我ñSCID小鼠的背部，使形成功能齐全，周细胞涂，混合型血管。我们的协议描述了如何制备含有人内皮有或没有人的周细胞，放置到SCID小鼠以及如何分析关键血管生成端点组织切片基质凝胶塞。

<table><tbody><tr><td>PBS (Phosphate buffered saline)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>0.25% Trypsin/0.53 mM EDTA</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Endothelial Cell Media (ECM) kit</td><td>Sciencell</td><td>1001</td><td>includes media, EC growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td></tr><tr><td>Pericyte Media (PM) kit</td><td>Sciencell</td><td>1201</td><td>includes media, Pericyte growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td></tr><tr><td>primary human pulmonary microvascular endothelial cells</td><td>Pulmonary Hypertension Breakthrough Initiative</td><td></td><td>this cell type is also available commercially. Cells used at passage 1-4</td></tr><tr><td>primary human pulmonary pericytes</td><td>Pulmonary Hypertension Breakthrough Initiative</td><td></td><td>this cell type is not available commercially, but brain paricytes are.&amp;nbsp; Cells used at passage 1-4</td></tr><tr><td>bFGF (basic Fibroblast Growth Factor)</td><td>Peprotech</td><td>100-18B</td><td>stock solution is 50 &amp;mu;g/ml in 0.1% BSA in PBS, aliquots at 50 &amp;mu;l and stored at -20 degrees</td></tr><tr><td>Matrigel Basement Membrane Matrix</td><td>BD</td><td>356237</td><td></td></tr><tr><td>28 G 1 cc Insluin Syringe</td><td>BD</td><td>329410</td><td></td></tr><tr><td>SCID (Severe Combined Immune Deficiency) mice</td><td>The Jackson Laboratory</td><td>5557</td><td>NOD.SCID IL2R gamma knockout strain is the best strain; 4-6 weeks of age</td></tr><tr><td>1.5 ml microcentrifuge tubes, sterile</td><td>Thermo Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>15 ml screw top tubes, sterile</td><td>BD Biosciences</td><td>352096</td><td></td></tr><tr><td>PAP pen</td><td>Life Technologies</td><td>8899</td><td></td></tr><tr><td>hemocytometer</td><td>ThermoFisher Scientific</td><td>02-671-6</td><td></td></tr><tr><td>Nair hair removal cream</td><td>Walmart</td><td></td><td></td></tr><tr><td>anti-human CD31 primary antibody</td><td>LifeSpan Biosciences</td><td>LS-B4737</td><td>working solution is 1:50</td></tr><tr><td>anti-human Smooth Muscle actin CY3 primary fluorescent antibody</td><td>Sigma</td><td>C6198</td><td>working solution is 1:300</td></tr><tr><td>goat anti-rabbit secondary antibody; 488/green</td><td>ThermoFisher Scientific</td><td>A-11008</td><td>working solution is 1:250</td></tr><tr><td>Prolong Gold DAPI solution</td><td>Cell Signaling</td><td>8961S</td><td></td></tr><tr><td>microscope slides</td><td>VWR</td><td>48300-047</td><td></td></tr><tr><td>no. 1.5 cover slips</td><td>Thermo Fisher Scientific</td><td>12-544-D</td><td></td></tr><tr><td>citrate buffer 10x</td><td>Millipore</td><td>21545</td><td></td></tr><tr><td>extra fine surgical scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Formalin (paraformaldehyde)</td><td>Thermo Fisher Scientific</td><td>245-685</td><td></td></tr><tr><td>Tissue cassettes</td><td>Simport</td><td>M492-12</td><td></td></tr><tr><td>goat serum</td><td>Dako</td><td>X0907</td><td></td></tr></tbody></table>

in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

基质凝胶塞段的代表性H＆E和免疫荧光染色， 如图2所示。节从EC仅插头显示一些血管被大多不与血液（ 图2左上，黑色箭头），而含有插头灌注既内皮和周显示多个灌注血管直径较大和完整周细胞包裹，就证明了紧邻的CD31阳性内皮细胞阳性的SMA染色。这些结果表明，周细胞中的新生血管形成，而这动态过程可以概括并容易在体内模型分析发挥重要作用。 <img alt="图1" src="/files/ftp_upload/54617/54617fig1.jpg" /> 图1.组织定位在组织盒嵌入式时。基质凝胶塞（黄色）分离后，它是在鼠标的肌肉（紫色）和皮肤（绿色）层之间。插头被放置在组织盒如图以便所有三个层可以在将第一切片中被切割的表面可以看到。 A）中查看从从磁带盒一侧的盒式磁带，B）查看的顶部。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54617/54617fig1large.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/54617/54617fig2.jpg" /> 图2.代表H＆E（上排）和基质凝胶的免疫荧光（下排）。图像显示内皮细胞光是外表（左图像）和ECS加上健康周细胞（PC）（右图像）。人和鼠内皮在绿色（底部图片）标记和周细胞（α平滑肌肌动蛋白（αSMA））被标记为红色（右下），和细胞核蓝染用DAPI染色。比例为25微米。这个数字已经从先前公布8修改。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54617/54617fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse at JoVE.com

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

我们提出了一个协议使用基质凝胶塞血管生成测定的变化来研究小鼠的人内皮周细胞的相互作用。

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

in-vivo-study-human-endothelial-pericyte-interaction-using-matrix-gel

Universidad San Sebastian

Research

JoVE Journal

Biology

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

9.6K 次观看.  Stanford University. 我们提出了一个协议使用基质凝胶塞血管生成测定的变化来研究小鼠的人内皮周细胞的相互作用。 

视频: In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

一个<em在体内</em分析测试血管通透性

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

科研

医学

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

免疫与感染

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

将毛细血管纳入内皮细胞珠发芽试验

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

发育生物学

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜微囊含量：血管生成在小鼠眼模型

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

神经科学

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

生物人类免疫缺陷小鼠的微血管网络

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

生物学

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

体外 使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

使用改良基质凝胶塞测定简化血管生成研究

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

作者聚焦：通过改良基质凝胶塞测定研究血管生成和血管通透性

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

用于血管生成研究 的改良体内 基质凝胶塞测定

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

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在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

将毛细血管纳入内皮细胞珠发芽试验

角膜微囊含量：血管生成在小鼠眼模型

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

生物人类免疫缺陷小鼠的微血管网络

评估血脑屏障网状血管形成和破坏的体外测定

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

体外使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

用于血管生成研究的改良体内基质凝胶塞测定

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

摘要

探索更多视频

此视频中的章节

一个

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

将毛细血管纳入内皮细胞珠发芽试验

角膜微囊含量：血管生成在小鼠眼模型

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

生物人类免疫缺陷小鼠的微血管网络

评估血脑屏障网状血管形成和破坏的体外测定

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

体外 使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

用于血管生成研究 的改良体内 基质凝胶塞测定

体外使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

用于血管生成研究的改良体内基质凝胶塞测定