Name: incorporating pericytes into an endothelial cell bead sprouting assay
Uploaded: 2018-02-16T15:00:00
Description: Watch this Scientific Journal Video about Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay at JoVE.com

我们感谢 Drs. 维多利亚 Bautch 和约书亚为帮助讨论和建议优化标准珠发芽试验条件和发芽化验染色协议。S.H.A. 得到了国家普通医学研究所授予 5T32 GM007092 奖的部分支持。

1天:
1. 血管内皮细胞的瞬态转染
<ol><li>如果需要, 使用基因调节寡核苷酸 (例如微 rna 或小干扰 RNA (siRNA)) 和适当的脂质体试剂, 对人脐静脉内皮细胞 (HUVEC) 进行瞬态转染, 并根据制造商的说明。 注: 该协议具有很大的成功执行反向 transfections 与定制 siRNA 序列和商业转染试剂 (见材料表)。作者没有测试其他转染的协议和试剂与此检测。使用所列试剂进行反?...

<ol>
	<li>Hanahan, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+and+emerging+mechanisms+of+the+angiogenic+switch+during+tumorigenesis.">Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.</a> Cell. 86 (3), 353-364 (1996).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+ischemic+and+neoplastic+disorders.">Angiogenesis in ischemic and neoplastic disorders.</a> Annu Rev Med. 54, 17-28 (2003).</li><li>Zhang, Z. G., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurorestorative+therapies+for+stroke:+underlying+mechanisms+and+translation+to+the+clinic.">Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic.</a> Lancet Neurol. 8 (5), 491-500 (2009).</li><li>Nakatsu, M. N., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+three-dimensional+in+vitro+model+for+the+analysis+of+angiogenesis.">An optimized three-dimensional in vitro model for the analysis of angiogenesis.</a> Methods Enzymol. 443, 65-82 (2008).</li><li>Nehls, V., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+microcarrier-based+in+vitro+assay+for+rapid+and+reliable+quantification+of+three-dimensional+cell+migration+and+angiogenesis.">A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis.</a> Microvasc Res. 50 (3), 311-322 (1995).</li><li>Sims, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pericyte--a+review.">The pericyte--a review.</a> Tissue Cell. 18 (2), 153-174 (1986).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Tattersall, I. W., 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=In+vitro+modeling+of+endothelial+interaction+with+macrophages+and+pericytes+demonstrates+Notch+signaling+function+in+the+vascular+microenvironment.">In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment.</a> Angiogenesis. 19 (2), 201-215 (2016).</li><li>Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+tumor+vasculature:+an+emerging+concept+in+antiangiogenic+therapy.">Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy.</a> Science. 307 (5706), 58-62 (2005).</li><li>Tahergorabi, Z., Khazaei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+angiogenesis+and+its+assays.">A review on angiogenesis and its assays.</a> Iran J Basic Med Sci. 15 (6), 1110-1126 (2012).</li><li>Popson, S. A., 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=Interferon-induced+transmembrane+protein+1+regulates+endothelial+lumen+formation+during+angiogenesis.">Interferon-induced transmembrane protein 1 regulates endothelial lumen formation during angiogenesis.</a> Arterioscler Thromb Vasc Biol. 34 (5), 1011-1019 (2014).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438 (7070), 937-945 (2005).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. 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该协议提出了一种方法, 以描述复杂的阶段和异型细胞相互作用的发芽血管生成通过使研究员使用遗传和成像方法进行彻底的机械调查。在进行检测时, 必须在珠搅拌步骤中进行有效的珠状血管内皮涂层。不良的内皮涂层将明显, 如果珠似乎没有像高尔夫球一样粗糙表面第二天早晨之前, 凝胶植入, 而不是完全平滑。注意确保在每一个搅拌步骤中的珠子充分悬浮, 通过大力搅拌管, 允许最大暴露的珠?...

血管生成对适当的胚胎发生和创面愈合至关重要, 在包括癌症进展1和冠状动脉疾病在内的众多疾病中也起着关键作用。2,3更好地了解在正常发育过程中血管生成的发生方式, 以及如何在病理环境中重新激活, 对于开发新颖有效的治疗方法至关重要。忠实的体外模型重述了血管生成体内中所涉及的重要阶段和细胞类型, 以使研究人员能够更好地描述推动血管生成的分子机制并作出新的发现。内皮细胞调节。
中津和休斯优化了发芽珠的检测, 他们已经证明经历了许多已知的阶段发芽血管生成。4,5本文提出的方法的目的是在中津和休斯通过将 perictyes 加入到实验中进行优化的分析, 从而将壁画细胞在内皮细胞发芽中的分泌和 juxtracrine 作用纳入新的血管生成研究。毛细血管是由它们的角色定义的壁画细胞, 因为它们被嵌入在血管基底膜中, 与内皮细胞保持紧密的物理接触。6毛细血管和内皮细胞通过信号通路进行复杂的交叉谈话, 包括凹口信号、Ang-Tie2、PDGFRβ、TGFRβ和许多其他。7,8小鼠模型在这些信号通路中缺乏, 表明在胚胎发生中发育血管的周细胞覆盖率较差, 导致血管重塑和功能失调。7此外, 毛细血管在病理血管生成中的作用是重要的, 但往往不受重视。例如, 肿瘤血管的一个独特的特点是, 血管是更不成熟, 漏水, 和功能失调, 由于不良的周细胞覆盖率。9已建议毛细血管的存在或缺失会显著影响肿瘤血管的表型, 是对血管生成和抗肿瘤治疗反应的重要调解人。9因此, 包括毛细血管在体外化验中的作用, 是更全面地捕捉内皮调节的重要机制的关键。
尽管目前有许多用于研究血管生成的体外和体内检测方法, 但在每个方面都存在一些不足之处。一些, 如内皮增生和内皮移植的检测, 是过度简化, 并集中在一个内皮功能的一个孤立的设置, 在组织培养塑料。10其他检测在3维 (3D) 设置中发生, 如胶管形成试验、10 , 但这些化验仍然过于简单化, 更注重内皮细胞迁移的能力, 并形成-新生血管结构, 而不是从预先存在的血管发芽。此外, 这些化验都没有纳入壁画细胞类型。有前体模型, 如环形主动脉检测, 包括毛细血管存在于宿主器官中, 但这些模型的遗传操作更具挑战性, 因为有必要产生挖空或转基因小鼠模型的感兴趣的途径。珠发芽法是理想的, 因为它模型内皮发芽, 增殖, 迁移, 甚至吻合和流明形成的3D 矩阵。4该检测忠实地允许对发芽的许多不同阶段进行机械性评估, 同时仍然允许在更受控制的环境中对内皮细胞或毛细血管进行直接的基因修饰。含有发芽珠的纤维蛋白凝块可以很容易地固定, 染色, 并在不同的发芽阶段成像;这些芽也可以放置在现场成像室, 以执行实时成像的发芽。本文提出的方法是研究血管生成的基本机制, 通过深入分型和深入分析血管生成过程中激活的通路。

<table>
	<tbody>
		<tr>
			<td>Sterile Pipette tips</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EGM2 Media Bullet Kit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>HUVEC Media</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Gibco</td>
			<td>11095114</td>
			<td>10T1/2 Media</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965118</td>
			<td>NHLF Media</td>
		</tr>
		<tr>
			<td>Tissue culture-grade PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Magnesium and calcium free</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>For lifting HUVEC</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>15050065</td>
			<td>For lifting 10T 1/2 and NHLF</td>
		</tr>
		<tr>
			<td>Customs siRNAs</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Life Technologies</td>
			<td>13778150</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>Lonza</td>
			<td>C2517A</td>
			<td></td>
		</tr>
		<tr>
			<td>10T 1/2</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NHLF</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytodex 3 microcarrier beads</td>
			<td>Sigma</td>
			<td>C3275</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture-coated 6 and 10 cm plates</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>t9549</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>a3428</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Tubes</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture incubator and hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well glass bottom plates</td>
			<td>MatTek</td>
			<td>P24G1.513F</td>
			<td>Glass-bottom plates are needed only if the sprouts are going to be imaged. If not, tissue culture plastic is also acceptable.</td>
		</tr>
		<tr>
			<td>Sterile Filtration Device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

incorporating pericytes into an endothelial cell bead sprouting assay

血管生成是新血管从预存在血管生长而来的, 是许多生物过程的重要组成部分, 包括胚胎发生和发育、伤口愈合、肿瘤生长和转移以及眼部和心血管疾病。有效的体外模型概括了血管生成的生物学, 需要适当地研究这一过程, 并确定可最终针对新的治疗策略的调控机制。微珠血管生成实验已被证明, 以概括的多阶段内皮发芽在体外.然而, 这一检测的局限性是缺乏内皮-壁画细胞相互作用,这是关键的分子和表型调节内皮细胞功能在体内。这里给出的协议提出了一个方法, 将壁画细胞纳入珠血管生成试验, 并表明在发芽体外的血管内皮细胞和毛细血管紧密联系。该议定书还详细说明了一种方法, 有效地沉默的目标基因使用 siRNA 在内皮细胞的机械学研究。总之, 本协议提供了一种体外分析方法, 可以更恰当地模拟发芽血管生成过程中所涉及的不同细胞类型, 并为治疗评估和新发现提供了一个更加生理学相关的平台。血管生成调控机制。

此协议允许两个单元格类型的紧密关联体外,和毛细血管的存在补充发芽的发生 (图 1A, B)。该协议还允许有效的沉默 (例如通过 RNA 干扰) 的基因感兴趣的细胞类型的兴趣 (如 VEGFA 特别是在内皮细胞或 PDGFRβ在毛细血管)7,12在检测期间, 这可转化为对发芽表型的抑制作用。使用本协议?...

Watch this Scientific Journal Video about Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay at JoVE.com

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

本协议提出了一种新的体外微珠分析方法, 更恰当地模拟体内的过程, 并结合毛细血管。这种修改使珠粒化验更忠实地重述内皮细胞和壁画细胞之间的异型细胞相互作用, 对血管生成至关重要。

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 ...

Research

JoVE Journal

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Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

内皮细胞迁移评估暴露于有毒化学品后，

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

科研

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Isolation of Murine Embryonic Hemogenic Endothelial Cells

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

无珠光高音喇叭在食子胚胎中的细胞力学探测

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

微流控法评价人诱导的多能干细胞内皮细胞 (hipsc-ec) 对白细胞粘附的评价

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...