Name: in vitro model of coronary angiogenesis
Uploaded: 2020-03-10T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Model of Coronary Angiogenesis at JoVE.com

作者感谢夏尔马实验室的成员提供了支持性的研究环境。我们特别感谢黛安（迪）R.霍夫曼谁维护和关心我们的老鼠群。我们还要感谢菲利普·斯马尔迪诺博士和卡罗琳·范恩博士对手稿进行彻底校对，并提出有益的评论。这项工作得到了鲍尔州立大学教务长办公室和生物学系对B.S、印第安纳科学院高级研究补助金基金和NIH（RO1-HL128503）和纽约干细胞基金会向K.R.提供资金的资助。

该协议中所有动物的使用遵循鲍尔州立大学机构动物护理和使用委员会（IACUC）的准则。
1. 建立小鼠饲养者和检测阴道塞的及时怀孕
<ol><li>建立一个与野生型雄性小鼠和雌性小鼠的小鼠繁殖笼。确保繁殖小鼠的年龄在6-8周之间。设置一对（1雄和1女）或作为三人（1雄和2雌）繁殖。</li>
	<li>第二天早上检查阴道塞。使用倾斜的金属探头通过将深塞插入阴道开口来检测深塞。指?...

<ol>
	<li>Red-Horse, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+arteries+form+by+developmental+reprogramming+of+venous+cells.">Coronary arteries form by developmental reprogramming of venous cells.</a> Nature. 464 (7288), 549-553 (2010).</li><li>Chen, H. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sinus+venosus+contributes+to+coronary+vasculature+through+VEGFC-stimulated+angiogenesis.">The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis.</a> Development. 141 (23), 4500-4512 (2014).</li><li>Volz, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+are+progenitors+for+coronary+artery+smooth+muscle.">Pericytes are progenitors for coronary artery smooth muscle.</a> Elife. 4, (2015).</li><li>Chen, H. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-C+and+aortic+cardiomyocytes+guide+coronary+artery+stem+development.">VEGF-C and aortic cardiomyocytes guide coronary artery stem development.</a> Journal of Clinical Investigation. 124 (11), 4899-4914 (2014).</li><li>Chang, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DACH1+stimulates+shear+stress-guided+endothelial+cell+migration+and+coronary+artery+growth+through+the+CXCL12-CXCR4+signaling+axis.">DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12-CXCR4 signaling axis.</a> Genes and Development. , (2017).</li><li>Tian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subepicardial+endothelial+cells+invade+the+embryonic+ventricle+wall+to+form+coronary+arteries.">Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries.</a> Cell Research. 23 (9), 1075-1090 (2013).</li><li>Wu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+Cells+Form+the+Coronary+Arteries+by+Angiogenesis+through+Myocardial-Endocardial+VEGF+Signaling.">Endocardial Cells Form the Coronary Arteries by Angiogenesis through Myocardial-Endocardial VEGF Signaling.</a> Cell. 151 (5), 1083-1096 (2012).</li><li>Katz, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+compartments+of+the+proepicardial+organ+give+rise+to+coronary+vascular+endothelial+cells.">Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells.</a> Developmental Cell. 22 (3), 639-650 (2012).</li><li>Das, S., Red-Horse, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+plasticity+in+cardiovascular+development+and+disease.">Cellular plasticity in cardiovascular development and disease.</a> Developmental Dynamics. 246 (4), 328-335 (2017).</li><li>Sharma, B., Chang, A., Red-Horse, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+Artery+Development:+Progenitor+Cells+and+Differentiation+Pathways.">Coronary Artery Development: Progenitor Cells and Differentiation Pathways.</a> Annual Review of Physiology. 79, 1-19 (2017).</li><li>Tian, X., Pu, W. T., Zhou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+origin+and+developmental+program+of+coronary+angiogenesis.">Cellular origin and developmental program of coronary angiogenesis.</a> Circulation Research. 116 (3), 515-530 (2015).</li><li>Wu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+cells+form+the+coronary+arteries+by+angiogenesis+through+myocardial-endocardial+VEGF+signaling.">Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling.</a> Cell. 151 (5), 1083-1096 (2012).</li><li>Gerhardt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+guides+angiogenic+sprouting+utilizing+endothelial+tip+cell+filopodia.">VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia.</a> Journal of Cell Biology. 161 (6), 1163-1177 (2003).</li><li>Ruhrberg, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+patterning+cues+provided+by+heparin-binding+VEGF-A+control+blood+vessel+branching+morphogenesis.">Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis.</a> Genes and Development. 16 (20), 2684-2698 (2002).</li><li>Kikuchi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+antiangiogenic+isoform+of+VEGF-A+contributes+to+impaired+vascularization+in+peripheral+artery+disease.">An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease.</a> Nature Medicine. 20 (12), 1464-1471 (2002).</li><li>Folkman, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+tumor+factor+responsible+for+angiogenesis.">Isolation of a tumor factor responsible for angiogenesis.</a> Journal of Experimental Medicine. 133 (2), 275-288 (1971).</li><li>Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+VEGF+in+the+regulation+of+physiological+and+pathological+angiogenesis.">The role of VEGF in the regulation of physiological and pathological angiogenesis.</a> Experientia Supplementum. (94), 209-231 (2005).</li><li>Ferrara, N., Bunting, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor,+a+specific+regulator+of+angiogenesis.">Vascular endothelial growth factor, a specific regulator of angiogenesis.</a> Current Opinion in Nephrology and Hypertension. 5 (1), 35-44 (1996).</li><li>Sharma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+Progenitor+Cells+Compensate+to+Rebuild+the+Coronary+Vasculature+in+Elabela-+and+Apj-Deficient+Hearts.">Alternative Progenitor Cells Compensate to Rebuild the Coronary Vasculature in Elabela- and Apj-Deficient Hearts.</a> Developmental Cell. 42 (6), 655-666 (2017).</li><li>Rhee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+deletion+of+Ino80+disrupts+coronary+angiogenesis+and+causes+congenital+heart+disease.">Endothelial deletion of Ino80 disrupts coronary angiogenesis and causes congenital heart disease.</a> Nature Communications. 9 (1), 368 (2018).</li></ol>

成功生长SV和内部原组织冠状血管的一些最关键的步骤是：1） 正确识别和隔离SV组织，用于SV培养;2） 使用e11~11.5年龄之间的胚胎心室，获得准确的恩多培养;3） 在整个解剖期间保持无菌状态，并始终保持组织冷;和4） 保持外植附在ECM涂层膜上，以避免组织漂浮在介质中。
首先，分离SV组织可能具有挑战性。重要的是要认识到，SV位于隐藏在肺卵管内的左右心的背侧。因此，很...

心脏的血管通常被称为冠状血管。这些血管由动脉、静脉和毛细血管组成。在开发过程中，先建立高度分支毛细血管，然后改造成冠状动脉和静脉1、2、3、4、5。这些初始毛细血管由在上皮管、正肠（SV）和内卡（Endo）组织1、6、7、8中发现的内皮祖细胞构建而成。SV是胚胎心脏的流入器官，内核是心脏流明的内衬。在SV和Endo中发现的内皮原细胞建立大部分的冠状血管，而外皮心肌贡献了相对小部分2。冠状血管毛细血管网络从先前存在的前体细胞在心脏中生长的过程称为冠状血管生成。冠状动脉疾病是全世界死亡的主要原因之一，然而，这种病却缺乏有效的治疗方法。了解冠状动脉生成的详细细胞和分子机制，对于设计修复和再生受损的冠状动脉的新颖有效的疗法非常有用。
最近，我们对冠状血管如何发展的认识激增，部分是通过开发新的工具和技术实现的。特别是，在体内系系标签和先进的成像技术在发现冠状血管9、10、11、12的细胞起源和分化途径方面非常有用。尽管这些在体内工具的优点，但在速度、灵活性和可访问性方面还是有局限性的。因此，强大的体外模型系统可以补充体内系统，以高通量的方式阐明冠状血管生成细胞和分子机制。
在这里，我们描述了冠状动脉成因的体外模型。我们开发了一种体外外培养系统，从两个祖组织SV和Endo生长冠状血管。通过这个模型，我们表明体外组织外植培养体生长冠状血管芽时，刺激生长介质。此外，在血管内皮生长因子A（VEGF-A）刺激时，外植培养物与对照生长迅速，这是一种高效力的血管生成蛋白。此外，我们发现，来自SV培养的血管生成芽经历静脉分化（COUP-TFII表达的丧失），这种机制类似于体内的SV血管生成。这些数据表明，体外外植物培养系统忠实地恢复了体内发生的血管生成事件。总之，此处描述的血管生成体外模型非常适合以高通量和可访问的方式探测冠状血管生成细胞和分子机制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 x 20 MM Tissue Culture Dish</td>
			<td>Fisher Scientific</td>
			<td>877222</td>
			<td>Referred in the protocol as Petri dish</td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Fisher Scientific</td>
			<td>08-772-51</td>
			<td></td>
		</tr>
		<tr>
			<td>8.0 uM PET membrane culture inserts</td>
			<td>Millipore Sigma</td>
			<td>MCEP24H48</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-rabbit 555</td>
			<td>Fisher Scientific</td>
			<td>A31572</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-rat 488</td>
			<td>Fisher Scientific</td>
			<td>A21206</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Angled Metal Probe</td>
			<td>Fine science tools</td>
			<td>10088-15</td>
			<td>Angled 45 degree, used for detecting deep plugs</td>
		</tr>
		<tr>
			<td>Anti- ERG 1/2/3 antibody</td>
			<td>Abcam</td>
			<td>Ab92513</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti- VE-Cadherin antibody</td>
			<td>Fisher Scientific</td>
			<td>BDB550548</td>
			<td>Primary antibody, manufacturer BD BioSciences</td>
		</tr>
		<tr>
			<td>CO2 gas tank</td>
			<td>Various suppliers</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Fisher Scientific</td>
			<td>13998223</td>
			<td>For 37 &#176;C, 5% CO2 incubation</td>
		</tr>
		<tr>
			<td>Dissection stereomicrosope</td>
			<td>Leica</td>
			<td>S9i</td>
			<td>Leica S9i Stereomicroscope</td>
		</tr>
		<tr>
			<td>EBM-2 basal media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td>Endothelial cell growth basal media</td>
		</tr>
		<tr>
			<td>ECM solution</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Commercially known as Matrigel</td>
		</tr>
		<tr>
			<td>EGM-2 MV Singlequots Kit</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td>Microvascular endothelial cell supplement kit; This is mixed into the EBM-2 to make the EGM-2 complete media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>SH3007003IR</td>
			<td></td>
		</tr>
		<tr>
			<td>FiJi</td>
			<td>NIH</td>
			<td>NA</td>
			<td>Image processing software (<a href="https://imagej.net/Fiji/Downloads" target="_blank">https://imagej.net/Fiji/Downloads</a>)</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine science tools</td>
			<td>11412-11</td>
			<td>Used for embryo dissection</td>
		</tr>
		<tr>
			<td>Fisherbrand Straight-Blade operating scissors</td>
			<td>Fisher Scientific</td>
			<td>13-808-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone Phosphate Buffered Saline (1X)</td>
			<td>Fisher Scientific</td>
			<td>SH-302-5601LR</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow tissue culture hood</td>
			<td>Fisher Scientific</td>
			<td>various models available</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Vectashield with DAPI</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy/Fisher</td>
			<td>50-980-494</td>
			<td>This is available at 32%; needs to be diluted to 4%</td>
		</tr>
		<tr>
			<td>Perforated spoon</td>
			<td>Fine science tools</td>
			<td>10370-18</td>
			<td>Useful in removing embryo/tissues from a solution</td>
		</tr>
		<tr>
			<td>Recombinant Murine VEGF-A 165</td>
			<td>PeproTech</td>
			<td>450-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard forceps, Dumont #5</td>
			<td>Fine science tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Sure-Seal Mouse/Rat chamber</td>
			<td>Easysysteminc</td>
			<td>EZ-1785</td>
			<td>Euthanasia chamber</td>
		</tr>
	</tbody>
</table>

in vitro model of coronary angiogenesis

在这里，我们描述了一个体外培养测定，以研究冠状血管生成。冠状血管喂养心肌，是临床上重要的。这些血管的缺陷代表严重的健康风险，如动脉粥样硬化，这可能导致心肌梗死和心脏衰竭的患者。因此，冠状动脉疾病是全世界死亡的主要原因之一。尽管它具有临床重要性，但如何再生受损的冠状动脉进展甚微。然而，最近在了解冠状血管发育的细胞起源和分化途径方面取得了进展。使研究人员能够荧光标记后代细胞、跟踪后代命运和想象体内后代的工具和技术的出现，有助于理解冠状血管的发展。体内研究很有价值，但在速度、可访问性和实验设计的灵活性方面却有局限性。或者，准确的冠状血管生成体外模型可以规避这些限制，使研究人员能够快速灵活地询问重要的生物学问题。缺乏适当的体外模型系统可能妨碍了理解冠状血管生长细胞和分子机制的进展。在这里，我们描述了一个体外培养系统，从心内膜（SV）和内膜（Endo）生长冠状血管，这是许多冠状血管产生的两个祖组织。我们还确认，这些文化准确地概括了一些已知的体内机制。例如，我们表明，SV培养中的血管生成芽与体内观察到的类似，对COUP-TFII的表达进行低调节。此外，我们显示VEGF-A，一个众所周知的血管生成因子在体内，有力地刺激血管生成从SV和Endo培养。总体而言，我们设计了一个精确的体外培养模型来研究冠状血管生成。

SV血管生成在体内最引人注目的特点之一是，它遵循一个特定的途径，涉及细胞去分化和再分化事件，发生在定型的时间和位置1。当最初的SV细胞生长到心脏心室时，它们停止产生静脉标记，如COUP-TFII（图7）。随后，冠状芽采取两种迁移路径，要么在心脏表面或心肌深处。水面船只最终成为静脉，而入侵的船只变成动脉和毛细血管<...

Watch this Scientific Journal Video about In Vitro Model of Coronary Angiogenesis at JoVE.com

In Vitro Model of Coronary Angiogenesis

冠状血管生成体外模型可用于发现冠状血管生成细胞和分子机制。正月线和内卡组织体外外培养物在响应VEGF-A时表现出强劲的生长，并表现出与体内类似的COUP-TFII表达模式。

冠状血管生成体模型

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

我们的协议很重要，因为它允许研究人员研究有机体外冠状血管生成分子机制。该技术的主要优点是，它准确地模拟了生物体内冠状动脉血管生成的过程，有助于研究冠状动脉发生机制。首先喷洒携带胚胎的怀孕小鼠11.5胚胎与70%乙醇。用钳子将皮肤举过腹部，用剪刀使腹部皮肤和腹腔切口横向和前到隔膜上露出子宫角。抓住子宫，切割组织自由，拉出子宫角。将胚胎串放入冷无菌PBS的立体显微镜下，从每个胚胎上剥离子宫肌肉、蛋黄囊和安尼翁盖。当每个胚胎被分离时，使用穿孔的勺子将组织转移到含有冰上无菌PBS的新培养皿中。要收获胚胎心脏组织，将第一个被解剖的胚胎移植到显微镜下的新培养皿中，然后将胚胎放在腹侧向上。使用细钳在胸部做一个小切口，略高于隔膜，并将封闭的钳子插入切口以扩大切口。使用钳子将胸壁打开以暴露心脏和肺部，使用第二对钳子轻轻移动心脏前 90 度，以暴露背动脉和静脉。然后，抓住心脏底部的这些血管，小心地拔出心脏和肺，然后用冷的PBS冲洗组织。当所有心脏组织都收获后，将心脏样本放在显微镜下，从每颗心脏中去除肺叶。将第一颗心放在其后侧，将心脏放在其底部，使用细钳从心脏中刮刮 SV 周围相邻组织中的左右角体。要隔离 SV，请将心后侧定向向上。小心地将 SV 从心脏中去除，并使用无菌转移移液器将分离的组织放入新的六厘米培养皿中，其中含有冰冷无菌 PBS。要隔离整个心室，请清除流出的管，并使用新的无菌转移移液器将心室放入一个单独的六厘米培养皿中，其中含有冰冷无菌 PBS。要建立SV和整个心室培养物，首先在24孔培养板的井中，先涂上一个8微米孔PET培养物的膜，每培养物100微升新鲜稀释的细胞外基质，在37摄氏度下至少30分钟。当基质凝固后，使用转移移液器将一个外植转移到每个刀片上，并在显微镜下移动板。使用清洁钳子，将外植物放在每个刀片的中心，以确保它们不会连接到侧壁上，并小心地移除任何额外的 PBS。接下来，将盖子关闭在层流组织培养罩下，将100微升预加热的完整介质添加到每个刀片，并在每个刀片下方的下面添加200微升。然后将300微升PBS添加到任何未使用的井中，并将板放入细胞培养箱5天。对于培养的第二天的VEGF-A治疗，从每种培养物的两个腔室中去除介质，并在每个刀片中加入100微升PBS，在每井中加入200微升PBS。旋转板几次后，用300微升基底介质补充1%的胎儿牛血清代替PBS，开始培养，然后将板返回孵化器。在饥饿后的第三天，在控制井中加入300微升新鲜基底培养基加血清，将每毫升VEGF-A的50毫微克添加到治疗井中，并将板回细胞培养箱。在培养的第六天，用PBS清洗房间，并修复培养物与200微升4%对甲醛在每个井，和100微升固定剂在每个插入。在 4 摄氏度下 20 分钟摇动后，用 300 微升 PBS 清洗培养物，每次洗涤 10 分钟，在室温下摇动。上次洗涤后，在井中加入300微升原抗体溶液，并在4摄氏度下插入一个带摇摆的过夜培养物。第二天早上，用摇动在PBS中用非离子表面活性剂清洗培养物10次，每10分钟更换一次洗涤溶液，然后用300微升的合适荧光结合二次抗体在4摄氏度过夜与摇动标记。第二天，用非离子表面活性剂在新鲜的PBS中清洗10次培养物，如所证明的。最后一次洗涤后，使用细钳小心地从每个刀片中剥离膜，并将膜放在单独的玻璃显微镜幻灯片上，将外植侧向上。用 DAPI 补充安装介质在盖滑中覆盖样品，用透明指甲油密封盖滑的边缘。指甲油干燥后，通过共和显微镜对幻灯片进行成像。当最初的SV细胞生长到心室时，它们停止产生静脉标记，如 Coup-TF2。经过五天的培养，SV内皮细胞发芽并迁移到膜上。与胚胎心脏一样，Coup-TF2表达随着容器从SV迁移而减少。VEGF-A的培养物在密度和长度上都增加了血管生成芽的生长。与对等对子相比，VEGF-A刺激的进一步SV培养表明芽长度增加了近三倍，这表明SV和内膜芽对VEGF-A有反应。重要的是不要失去SV组织，同时拉出心脏和肺，去除牙底，并清洁SV周围的相邻组织。可以进行实时成像实验，以可视化冠状动脉血管生成，并在这个过程中捕捉细胞和分子动力学的细节。这项技术对于评估冠心血管生成的潜在分子机制，作为一般血管生成研究的模型系统，是非常有用的。

Research

JoVE Journal

Medicine

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

结扎冠状动脉内注射在小鼠模型梗死

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

科研

医学

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

一个小鼠模型角膜血管生成研究掌上含量

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Angiogenesis in the Ischemic Rat Lung

在缺血大鼠肺的血管生成

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

猪胫骨移植术后血管生成的动物模型

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

小鼠外周动脉疾病模型中单核细胞归巢和肿瘤相关血管生成的活体显微镜观察

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...