Name: a patient-derived xenograft model for venous malformation
Uploaded: 2020-06-15T15:00:00
Description: Watch this Scientific Journal Video about A Patient-Derived Xenograft Model for Venous Malformation at JoVE.com

作者要感谢诺拉湖校对。本手稿中报告的研究得到了国家心脏、肺和血液研究所的支持，该学会获得国家健康研究院的一部分，其奖项编号为 R01 HL117952（E.B.）。内容完全由作者负责，不一定代表国家卫生研究院的官方观点。

患者组织样本是在辛辛那提儿童医院医疗中心（CCHMC）、癌症和血液疾病研究所根据机构政策经批准的机构审查委员会（IRB）获得组织样本和肿瘤和血管异常患者组织样本和数据收集和储存的知情同意后，经临床调查委员会批准的。以下所述的所有动物程序都经过 CCHMC 机构动物护理和使用委员会的审查和批准。
1. 材料和库存解决方案的准备
<ol><li>完整内皮细胞生长介质的准...

<ol>
	<li>Dompmartin, A., Vikkula, M., Boon, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venous+malformation:+update+on+aetiopathogenesis,+diagnosis+and+management.">Venous malformation: update on aetiopathogenesis, diagnosis and management.</a> Phlebology: The Journal of Venous Disease. 25 (5), 224-235 (2010).</li><li>Limaye, N., 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=Somatic+mutations+in+angiopoietin+receptor+gene+TEK+cause+solitary+and+multiple+sporadic+venous+malformations.">Somatic mutations in angiopoietin receptor gene TEK cause solitary and multiple sporadic venous malformations.</a> Nature Genetics. 41 (1), 118-124 (2009).</li><li>Castel, P., 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=Somatic+PIK3CA+mutations+as+a+driver+of+sporadic+venous+malformations.">Somatic PIK3CA mutations as a driver of sporadic venous malformations.</a> Science Translational Medicine. 8 (332), 42 (2016).</li><li>Limaye, N., 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=Somatic+Activating+PIK3CA+Mutations+Cause+Venous+Malformation.">Somatic Activating PIK3CA Mutations Cause Venous Malformation.</a> The American Journal of Human Genetics. 97 (6), 914-921 (2015).</li><li>Castillo, S. D., 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=Somatic+activating+mutations+in+Pik3ca+cause+sporadic+venous+malformations+in+mice+and+humans.">Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans.</a> Science Translational Medicine. 8 (332), 43 (2016).</li><li>Stratman, A. N., 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+cell+lumen+and+vascular+guidance+tunnel+formation+requires+MT1-MMP-dependent+proteolysis+in+3-dimensional+collagen+matrices.">Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP-dependent proteolysis in 3-dimensional collagen matrices.</a> Blood. 114 (2), 237-247 (2009).</li><li>Okada, S., Vaeteewoottacharn, K., Kariya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Highly+Immunocompromised+Mice+for+the+Establishment+of+Patient-Derived+Xenograft+(PDX)+Models.">Application of Highly Immunocompromised Mice for the Establishment of Patient-Derived Xenograft (PDX) Models.</a> Cells. 8 (8), 889 (2019).</li><li>Byrne, A. T., 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=Interrogating+open+issues+in+cancer+precision+medicine+with+patient-derived+xenografts.">Interrogating open issues in cancer precision medicine with patient-derived xenografts.</a> Nature Reviews Cancer. 17 (4), 254-268 (2017).</li><li>Allen, P., Melero-Martin, J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+collagen,+fibrin+and+PuraMatrix+matrices+provide+permissive+environments+for+human+endothelial+and+mesenchymal+progenitor+cells+to+form+neovascular+networks.">Type I collagen, fibrin and PuraMatrix matrices provide permissive environments for human endothelial and mesenchymal progenitor cells to form neovascular networks.</a> Journal of Tissue Engineering and Regenerative Medicine. 5 (4), 74 (2011).</li><li>Allen, P., Kang, K. T., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+onset+of+perfused+blood+vessels+after+implantation+of+ECFCs+and+MPCs+in+collagen,+PuraMatrix+and+fibrin+provisional+matrices.">Rapid onset of perfused blood vessels after implantation of ECFCs and MPCs in collagen, PuraMatrix and fibrin provisional matrices.</a> Journal of Tissue Engineering and Regenerative. 9 (5), 632-636 (2015).</li><li>Nowak-Sliwinska, P., 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=Consensus+guidelines+for+the+use+and+interpretation+of+angiogenesis+assays.">Consensus guidelines for the use and interpretation of angiogenesis assays.</a> Angiogenesis. 21 (3), 425 (2018).</li><li>Roh, Y. N., 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+results+of+surgical+treatment+for+patients+with+venous+malformations.">The results of surgical treatment for patients with venous malformations.</a> Annals of Vascular Surgery. 26 (5), 665-673 (2012).</li><li>Marler, J. J., Mulliken, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+management+of+hemangiomas+and+vascular+malformations.">Current management of hemangiomas and vascular malformations.</a> Clinics in Plastic Surgery. 32 (1), 99-116 (2005).</li><li>Goines, 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=A+xenograft+model+for+venous+malformation.">A xenograft model for venous malformation.</a> Angiogenesis. 21 (4), 725-735 (2018).</li><li>Li, 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=Ponatinib+Combined+With+Rapamycin+Causes+Regression+of+Murine+Venous+Malformation.">Ponatinib Combined With Rapamycin Causes Regression of Murine Venous Malformation.</a> Arteriosclerosis, thrombosis, and vascular biology. 39 (3), 496-512 (2019).</li><li>Le Cras, T. D., 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=Constitutively+active+PIK3CA+mutations+are+expressed+by+lymphatic+and+vascular+endothelial+cells+in+capillary+lymphatic+venous+malformation.">Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation.</a> Angiogenesis. , 1-18 (2020).</li><li>Boscolo, E., 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=Rapamycin+improves+TIE2-mutated+venous+malformation+in+murine+model+and+human+subjects.">Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects.</a> Journal of Clinical Investigation. 125 (9), 3491-3504 (2015).</li></ol>

在这里，我们描述了一种生成患者派生的 VM 异种移植模型的方法。这个 Murine 模型提供了一个优秀的系统，使研究人员能够更深入地了解病理流明的增大，并将有助于开发更有效和有针对性的治疗 VM。这可以很容易地适应调查其他类型的血管异常，如毛细血管淋巴静脉畸形16。有几个步骤对于成功生成可重复的血管病变至关重要。首先，患者衍生的内皮细胞必须是纯的（没有其?...

血管发育中的缺陷是许多疾病的根本原因，包括静脉畸形（VM）。VM是一种先天性疾病，其特征是血管异常形态和扩张。关于VM组织和内皮细胞（EC）的重要研究已经确定了两个基因的功能增益突变：TEK，编码酪氨酸激酶受体TYE2，和PIK3CA，编码PI3-激酶（PI3K）的p110+（催化亚基）等构形式2,32，3，4，5。,4,5这些体细胞突变导致关键血管生成/生长信号通路（包括PI3K/AKT）的配体独立超活化，从而导致扩张的肠黄静脉3。尽管这些重要的基因发现，随后的细胞和分子机制触发异常血管生成和扩大血管通道的形成仍然没有完全了解。
在正常和病理血管生成期间，新血管从预先存在的血管网络发芽，EC经历一系列重要的细胞过程，包括增殖、迁移、细胞外基质（ECM）重塑和流明形成6。EC 的二维和三维 （2D/3D） 体外培养物是单独研究每个细胞特性的重要工具。然而，显然需要用鼠标模型在宿主微环境内重述病理血管的扩大，同时为转化研究的目标药物提供有效的临床前评估平台。
到目前为止，尚未报告与 TIE2 功能增益突变相关的 VM 转基因喃生基因模型。目前的转基因VM小鼠模型依赖于激活突变PIK3CA p.H1047R3，5的无所不在或组织受限的表达。这些转基因动物提供了对这个热点PIK3CA突变的全身或组织特异性效应的显著洞察。这些模型的局限性是形成高度病理的血管网络，导致早期杀伤力。因此，这些小鼠模型不能充分反映突变事件的零星发生和VM病理学的局部性质。
相反，患者衍生的异种移植模型是基于移植或注射病理组织或细胞从患者衍生到免疫缺陷小鼠7。Xenograft模型是一个强大的工具，以扩大有关疾病开发和发现新的治疗剂8的知识。此外，使用患者衍生细胞允许科学家重新综述突变异质性，以研究患者表型的光谱。
在这里，我们描述了一个协议，其中患者衍生的VM EC，表示一个突变的组成活性形式的 TIE2 和/或PIK3CA被注射皮下在脂肪裸鼠的背部。注射的血管细胞悬浮在ECM框架中，以促进血管生成，如以前的血管异种移植模型9，10，1110,所述。9这些VM EC经历显著形态生成，在没有支撑细胞的情况下产生扩大的、充满的病理血管。VM 的异种移植模型为靶向药物的临床前评估提供了一个有效的平台，用于抑制不受控制的流明扩张的能力。

<table>
	<tbody>
		<tr>
			<td>Athymic nude mice, (Foxn1-nu); 5-6 weeks, males</td>
			<td>Envigo</td>
			<td>069(nu)/070(nu/+)</td>
			<td>Subcutaneous injection</td>
		</tr>
		<tr>
			<td>Biotinylated Ulex europeaus Agglutinin-I (UEA-I)</td>
			<td>Vector Laboratories</td>
			<td>B-1065</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Bottle top filter (500 ml; 0.2 &#181;M)</td>
			<td>Thermo Fisher</td>
			<td>974106</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>BSA</td>
			<td>A7906-50MG</td>
			<td>Cell culture; Histological analysis</td>
		</tr>
		<tr>
			<td>Calcium cloride dihydrate (CaCl2.2H2O)</td>
			<td>Sigma</td>
			<td>C7902-500G</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Caliper</td>
			<td>Electron Microscopy Sciences</td>
			<td>50996491</td>
			<td>Lesion plug measurment</td>
		</tr>
		<tr>
			<td>CD31-conjugated magnetic beads (Dynabeads)</td>
			<td>Life Technologies</td>
			<td>11155D</td>
			<td>EC separation</td>
		</tr>
		<tr>
			<td>Cell strainer (100 &#956;M)</td>
			<td>Greiner</td>
			<td>542000</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10103578001</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Conical Tube; polypropylene (15 mL)</td>
			<td>Greiner</td>
			<td>07 000 241</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Conical Tube; polypropylene (50 mL)</td>
			<td>Greiner</td>
			<td>07 000 239</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Coplin staining jar</td>
			<td>Ted Pella</td>
			<td>21029</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Coverglass (50 X 22 mm)</td>
			<td>Fisher Scientific</td>
			<td>12545E</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>DAB: 3,3&#39;Diaminobenzidine Reagent (ImmPACT DAB)</td>
			<td>Vector Laboratories</td>
			<td>SK-4105</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-027-CV</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DynaMag-2</td>
			<td>Life Technologies</td>
			<td>12321D</td>
			<td>EC separation</td>
		</tr>
		<tr>
			<td>Ear punch</td>
			<td>VWR</td>
			<td>10806-286</td>
			<td>Subcutaneous injection</td>
		</tr>
		<tr>
			<td>EDTA (0.5M, pH 8.0)</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium-2 (EGM2) Bulletkit (basal medium and supplements)</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Eosin Y (alcohol-based)</td>
			<td>Thermo Scientific</td>
			<td>71211</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS) , HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30910.03</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Filter tip 1,250 &#956;L</td>
			<td>MidSci</td>
			<td>AV1250-H</td>
			<td>Multiple steps</td>
		</tr>
		<tr>
			<td>Filter tip 20 &#956;L</td>
			<td>VWR</td>
			<td>10017-064</td>
			<td>Multiple steps</td>
		</tr>
		<tr>
			<td>Filter tip 200 &#956;L</td>
			<td>VWR</td>
			<td>10017-068</td>
			<td>Multiple steps</td>
		</tr>
		<tr>
			<td>Formalin buffered solution (10%)</td>
			<td>Sigma</td>
			<td>F04586</td>
			<td>Lesion plug dissection</td>
		</tr>
		<tr>
			<td>Hemacytometer (INCYTO; Disposable)</td>
			<td>SKC FILMS</td>
			<td>DHCN015</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Vector Hematoxylin</td>
			<td>H-3401</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Human plasma fibronectin purified protein (1mg/mL)</td>
			<td>Sigma</td>
			<td>FC010-10MG</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide solution (30% w/w)</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td></td>
			<td></td>
			<td>Analysis</td>
		</tr>
		<tr>
			<td>Isoflurane, USP</td>
			<td>Akorn Animal Health</td>
			<td>59399-106-01</td>
			<td>Subcutaneous injection</td>
		</tr>
		<tr>
			<td>magnesium sulfate heptahydrate (MgSO4.7H2O)</td>
			<td>Sigma</td>
			<td>M1880-500G</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Basement Membrane Matrix (Phenol Red-Free; LDEV-free)</td>
			<td>Corning</td>
			<td>356237</td>
			<td>Subcutaneous injection</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube (1.5 mL)</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td>EC separation</td>
		</tr>
		<tr>
			<td>Microscope Slide Superfrost (75mm X 25mm)</td>
			<td>Fisher Scientific</td>
			<td>1255015-CS</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Needles, 26G x 5/8 inch Sub-Q sterile needles</td>
			<td>Becton Dickinson (BD)</td>
			<td>BD305115</td>
			<td>Subcutaneous injection</td>
		</tr>
		<tr>
			<td>Normal horse serum</td>
			<td>Vector Laboratories</td>
			<td>S-2000</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-L-Glutamine (100X)</td>
			<td>Corning</td>
			<td>30-009-CI</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Permanent mounting medium (VectaMount)</td>
			<td>Vector Laboratories</td>
			<td>H-5000</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Pestle Size C, Plain</td>
			<td>Thomas Scientific</td>
			<td>3431F55</td>
			<td>EC isolation</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>VWR</td>
			<td>65500-202</td>
			<td>Subcutaneous injection</td>
		</tr>
		<tr>
			<td>Serological pipettes (10 ml)</td>
			<td>VWR</td>
			<td>89130-898</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Serological pipettes (5ml)</td>
			<td>VWR</td>
			<td>89130-896</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma</td>
			<td>223530</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Streptavidin, Horseradish Peroxidase, Concentrate, for IHC</td>
			<td>Vector Laboratories</td>
			<td>SA-5004</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Syringe (60ml)</td>
			<td>BD Biosciences</td>
			<td>309653</td>
			<td>Cel culture</td>
		</tr>
		<tr>
			<td>SYRINGE FILTER (0.2 &#181;M)</td>
			<td>Corning</td>
			<td>431219</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Syringes (1 mL with Luer Lock)</td>
			<td>Becton Dickinson (BD)</td>
			<td>BD-309628</td>
			<td>Subcutaneous injection</td>
		</tr>
		<tr>
			<td>Tissue culture-treated plate (100 X 20 mm)</td>
			<td>Greiner</td>
			<td>664160</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Tissue culture-treated plate (145X20 mm)</td>
			<td>Greiner</td>
			<td>639160</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Tissue culture-treated plates (60 X 15) mm</td>
			<td>Eppendorf</td>
			<td>30701119</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Tris-base (Trizma base)</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution (0.4 %)</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Trypsin EDTA, 1X (0.05% Trypsin/0.53mM EDTA)</td>
			<td>Corning</td>
			<td>25-052-Cl</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Biorad</td>
			<td>170-6531</td>
			<td>Histological anlaysis</td>
		</tr>
		<tr>
			<td>Wheaton bottle</td>
			<td>VWR</td>
			<td>16159-798</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Fisher Scientific</td>
			<td>X3P-1GAL</td>
			<td>Histological anlaysis</td>
		</tr>
	</tbody>
</table>

a patient-derived xenograft model for venous malformation

静脉畸形 （VM） 是一种血管异常，由静脉网络发育受损而引起，导致静脉扩张且经常功能失调。本文的目的是仔细描述一个模拟人类VM并且能够反映患者异质性的Murine异种移植模型的建立。在内皮细胞（EC）中超活化非遗传（体细胞 ）TEK（TIE2） 和 PIK3CA 突变被确定为VM中病理血管增大的主要驱动因素。以下协议描述了患者衍生的EC表达突变 TIE2 和/或 PIK3CA 的隔离、纯化和扩展。这些EC被注射到免疫缺陷的脑膜小鼠的背部，以产生分血管通道。TIE2 或 PIK3CA 突变 EC 产生的病变在注射后 7~u20129 天内明显血管化，并重述 VM 患者组织组织组织组织病理学特征。此 VM 异种移植模型为研究驱动 VM 形成和扩展的细胞和分子机制提供了一个可靠的平台。此外，该模型将有助于转化研究，测试新药候选药物在防止人类VM中异常血管增大的疗效。

该协议描述了基于皮肤注射患者衍生的EC到免疫缺陷裸鼠背部的VM的murine异种移植模型的过程。内皮细胞菌落可以在从VM组织或病变血中分离后4周内收获（图1A，B）。注射后的第二天，异种移植病变塞覆盖面积约80\u2012100 mm2。在我们的手中，带 TIE2/PIK3CA 突变EC的病变塞在注射14、15（图1C-E）后7~u20129天内明显15血...

Watch this Scientific Journal Video about A Patient-Derived Xenograft Model for Venous Malformation at JoVE.com

A Patient-Derived Xenograft Model for Venous Malformation

我们提出了一个详细的协议，以生成静脉畸形的杂音异种模型。该模型基于患者衍生的内皮细胞的皮下注射，这些内皮细胞含有超激活的 TIE2 和/或 PIK3CA 基因突变。异种移植病变密切回顾VM患者组织组织组织病理学特征。

静脉畸形的患者衍生异种移植模型

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

该协议可用于创建患者衍生的异种移植，重新概括静脉畸形的异质体。这进一步被允许进行我们的临床前治疗测试。该技术提供了一个快速可靠的NVivo系统，允许每天监测患者直接内皮细胞的生长和对实验治疗的反应。这种方法可以很容易地适应研究其他类型的血管或淋巴异常。首先准备注射细胞悬浮液。在37摄氏度下用5毫升预加热0.05%的edin-EDTA对内皮细胞进行试穿，两分钟。中和行程，加入五毫升EGM，将细胞收集到150毫升锥形管中。将其离心至400倍G5分钟，然后吸升，然后用三毫升EGM重新暂停细胞。用血细胞计计算细胞。将所需细胞号的体积冒入新的50毫升锥形管中，通过离心再次对细胞进行颗粒状。吸上液留下一个小体积来松开细胞颗粒。每次注射用220微升BMEM重新注入细胞颗粒。将细胞悬浮液完全混合在冰上，确保避免产生气泡。通过拉动柱塞将 BMEM 细胞培养混合物吸入一毫升注射器开口。您将将 26 量无菌针头锁定在注射器上，并在注射前将准备好的注射器平放在冰上。在确保小鼠正确麻醉后，将其放在胃部，用70%乙醇对注射区域进行消毒。轻轻滚动准备好的注射器，以重新注入任何已结算的细胞。将气泡剥到注射器的末尾，并排出一小卷细胞悬浮液。在注射部位捏皮，形成像结构一样帐篷。然后将针头插入皮肤下，通过释放捏皮防止注射到肌肉组织，确保针头只是皮肤深。持针稳稳地小心地注射200微升的细胞悬浮液，形成小球形质量。注意避免将细胞悬浮液注射到肌肉中。使用卡钳测量每个插头的长度和宽度。将解剖的异种移植插头对齐在尺子旁边的切割板上，并拍摄图像以记录病变的血管总。然后通过将插头淹没在室温下过夜 10% 的甲醛来固定插头。解剖后，病变塞被处理进行组织学分析，并染色为UEA-1检测插头内的人类衍生内皮细胞。在病变部分内的 X 平面图案中，用明亮的场显微镜在 20 X 放大时拍摄每个病变部分的 5 张 HPF 图像，以避免重叠。请确保在拍摄的图像上包含比例条。打开图像中的 HPF 图像J.To校准比例栏的像素，使用直线工具并查看比例图条。然后，通过单击"分析"和"设置比例"将测量的像素转换为毫米。接下来单击"分析集测量"并选择区域并添加到叠加。使用分析和测量来测量 HPF 的总字段面积，从而保存此测量值以进行定量测量。使用自由手选择工具手动勾勒出 UEA-1 正血管通道。单击"分析和测量"以量化概述区域。测量 HPF 内的所有血管通道。对每个部分中采取的五个 HPF 重复此过程，然后将值转移到 Excel 以进行进一步分析。然后根据手稿方向计算血管面积和血管密度的百分比。使用此协议，TIE2 或 PIK3CA 多活性突变内皮细胞的病变塞在注射后 7 至 9 天内明显血管化并扩散。病变塞与人体VM组织特征相似，由一层薄薄的内皮细胞对齐的大血管是可见的。这些血管结构通常含有红细胞，确认功能性脑膜病与宿主小鼠血管。使用人类特异性卵石UEA-1的免疫组织化学染色证实，血管区域内衬细胞来自人类植入细胞，而不是小鼠血管。按照这个程序，可以执行增殖或凋亡标记的病变部分的额外染色，以分析实验治疗的具体效果。此造化模型已用于静脉畸形新疗法的临床前研究，如 mTOR 抑制剂拉帕霉素，在 VM 患者的多项临床试验中显示出疗效。

Research

JoVE Journal

Developmental Biology

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

建立临床相关<em&gt;离体</em&gt;调查上皮创伤修复的微环境本地人模拟白内障手术模型

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

科研

发育生物学

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

小鼠胚胎跖骨文化：软骨内骨化的生理模型

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

用小鼠模型对精子发生细胞学分析的精管壁球技术

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

部分小叶切除术: 形态学肝再生的外科模型

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

A Mouse Distraction Osteogenesis Model

小鼠牵张成骨模型

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...