Name: a comprehensive procedure to evaluate the in vitro performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay
Uploaded: 2018-04-12T14:15:00
Description: Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay at JoVE.

这项工作得到了上海科学技术委员会 (15411951800, 15410723200) 的资助。作者感谢复旦大学病原微生物系玉梅教授和赵教授的技术支持。

该方法是根据复旦大学华山医院研究伦理学委员会批准的指导方针和规定进行的。每个步骤都遵循相应的标准安全措施。有关示意性演示文稿, 请参阅图 1。
1. 细胞培养和质粒结构
<ol><li>例行维持人脐静脉内皮细胞 (HUVEC) 在 Dulbecco 的改良鹰的培养基 (DMEM) 补充10% 胎牛血清 (血清), 100 单位/毫升青霉素, 100 µg/毫升链霉素在加湿 5% CO2孵化器摄氏37摄氏度。</li...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+capillary+haemangioblastoma:+the+pathologist's+viewpoint.">Central nervous system capillary haemangioblastoma: the pathologist's viewpoint.</a> Int J Exp Pathol. 88 (5), 311-324 (2007).</li><li>Ma, 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=Hemangioblastomas+might+derive+from+neoplastic+transformation+of+neural+stem+cells/progenitors+in+the+specific+niche.">Hemangioblastomas might derive from neoplastic transformation of neural stem cells/progenitors in the specific niche.</a> Carcinogenesis. 32 (1), 102-109 (2011).</li><li>Zhuang, Z., 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=Tumor+derived+vasculogenesis+in+von+Hippel-Lindau+disease-associated+tumors.">Tumor derived vasculogenesis in von Hippel-Lindau disease-associated tumors.</a> Sci Rep. 4, 4102 (2014).</li><li>Glasker, 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=VHL-deficient+vasculogenesis+in+hemangioblastoma.">VHL-deficient vasculogenesis in hemangioblastoma.</a> Exp Mol Pathol. 96 (2), 162-167 (2014).</li><li>Wizigmann-Voos, S., Breier, G., Risau, W., Plate, K. 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H., Kamijyo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+hemangioblastoma:+histogenesis+of+stroma+cells.">Cerebellar hemangioblastoma: histogenesis of stroma cells.</a> Cancer. 31 (6), 1528-1540 (1973).</li><li>Jurco, 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=Hemangioblastomas:+histogenesis+of+the+stromal+cell+studied+by+immunocytochemistry.">Hemangioblastomas: histogenesis of the stromal cell studied by immunocytochemistry.</a> Hum Pathol. 13 (1), 13-18 (1982).</li><li>Ma, 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=Identification+of+tumorigenic+cells+and+implication+of+their+aberrant+differentiation+in+human+hemangioblastomas.">Identification of tumorigenic cells and implication of their aberrant differentiation in human hemangioblastomas.</a> Cancer Biol Ther. 12 (8), 727-736 (2011).</li><li>Ma, 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=CD41+and+CD45+expression+marks+the+angioformative+initiation+of+neovascularisation+in+human+haemangioblastoma.">CD41 and CD45 expression marks the angioformative initiation of neovascularisation in human haemangioblastoma.</a> Tumour Biol. 37 (3), 3765-3774 (2016).</li><li>Sharifpanah, F., Sauer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Spheroid-Based+Sprout+Assay+in+Three-Dimensional+Fibrin+Scaffold:+A+Novel+In+Vitro+Model+for+the+Study+of+Angiogenesis.">Stem Cell Spheroid-Based Sprout Assay in Three-Dimensional Fibrin Scaffold: A Novel In Vitro Model for the Study of Angiogenesis.</a> Methods Mol Biol. 1430, 179-189 (2016).</li><li>Cai, 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=Long+non-coding+RNA+taurine+upregulated+1+enhances+tumor-induced+angiogenesis+through+inhibiting+microRNA-299+in+human+glioblastoma.">Long non-coding RNA taurine upregulated 1 enhances tumor-induced angiogenesis through inhibiting microRNA-299 in human glioblastoma.</a> Oncogene. 36 (3), 318-331 (2017).</li><li>Xu, 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=Construction+of+Conveniently+Screening+pLKO.1-TRC+Vector+Tagged+with+TurboGFP.">Construction of Conveniently Screening pLKO.1-TRC Vector Tagged with TurboGFP.</a> Appl Biochem Biotechnol. 181 (2), 699-709 (2017).</li><li>Laib, A. M., 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=Spheroid-based+human+endothelial+cell+microvessel+formation+in+vivo.">Spheroid-based human endothelial cell microvessel formation in vivo.</a> Nat Protoc. 4 (8), 1202-1215 (2009).</li><li>D'Alessio, A., Moccia, F., Li, J. H., Micera, A., Kyriakides, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+Vasculogenesis+in+Health+and+Disease.">Angiogenesis and Vasculogenesis in Health and Disease.</a> Biomed Res Int. 2015, 126582 (2015).</li><li>Finkenzeller, G., Graner, S., Kirkpatrick, C. J., Fuchs, S., Stark, G. 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T. <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+models+of+angiogenesis+and+vasculogenesis+in+fibrin+gel.">In vitro models of angiogenesis and vasculogenesis in fibrin gel.</a> Exp Cell Res. 319 (16), 2409-2417 (2013).</li><li>Blacher, 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=Cell+invasion+in+the+spheroid+sprouting+assay:+a+spatial+organisation+analysis+adaptable+to+cell+behaviour.">Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour.</a> PLoS One. 9 (5), 97019 (2014).</li><li>Straume, O., 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=Suppression+of+heat+shock+protein+27+induces+long-term+dormancy+in+human+breast+cancer.">Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer.</a> Proc Natl Acad Sci U S A. 109 (22), 8699-8704 (2012).</li><li>Naumov, G. N., Akslen, L. A., 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=Role+of+angiogenesis+in+human+tumor+dormancy:+animal+models+of+the+angiogenic+switch.">Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch.</a> Cell Cycle. 5 (16), 1779-1787 (2006).</li><li>Naumov, G. 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近年来, 血管内皮细胞15的研究刺激了多领域的维管束生物学研究。本文以内皮球发芽技术为实验模型, 研究了 VHL 基因在操作内皮细胞中的功能丧失对血管形成的作用, 以确定新的候选分子血管生成叶。根据我们的知识, 这是第一个检查内生改良内皮细胞血管生成效应的报告。
组织 (包括肿瘤组织) 新生血管是一个复杂的过程, 发生在发育过程中的通过?...

小脑 (哈佛商学院) 是良性血管肿瘤, 是完全在人类中枢神经系统 (CNS) 发现。他们发展在患者与冯冯·希佩尔·林道-林道 (VHL) 疾病或零星的损伤。VHL-哈佛商学院是难以治愈的外科治疗, 由于频繁复发和多病变, 造成这种遗传紊乱1。尽管 VHL 抑癌基因的失活已被认为是 VHL-哈佛商学院肿瘤发生的根本原因, 但哈佛商学院的细胞学起源 (包括新生血管) 和进化过程仍然很有争议2。因此, 更好地了解 HB-新生血管的生物机制, 可以为 VHL-哈佛商学院最有前途的抗血管策略提供有益的见解。
最近的研究表明, HB-新生血管类似于胚胎血管发生 3, 4, 5.典型的血管内皮生长因子 (VEGF) 介导的血管生成是由血管内皮引起的, 由 VHL 功能丧失所驱动, 导致增殖和新生血管形成, 这已被挑战6。在 1965年, Cancilla 和齐默尔曼使用电子显微镜发现, 哈佛商学院起源于内皮细胞7。后来发现基质细胞来源于 vasoformative 元素8。在 1982年, Jurco et 等发现基质细胞是内皮源9。因此, 我们假设, 人血管内皮细胞是血红蛋白的原细胞-新生血管的10。虽然最好使用从 VHL 患者手术中提取的 hb 细胞中的主要培养物, 但我们以前的研究表明, 来自 hb 的主要培养基不稳定, 细胞系无法建立3。此外, 3D 环境中的主要文化无法识别乙肝新生血管的细胞学来源, 因为它们包括 hb-血管成分的祖细胞10,11。因此, 作为一种原始和经典的内皮细胞模型, 人血管内皮细胞 (HUVEC) 可以作为哈佛商学院的替代细胞模型。
球形发芽试验是组织工程中的一个新模型12,13。本文开发了一种基于3D 胶原的共培养系统体外, 其目的是评价哈佛商学院是否存在经典的肿瘤血管生成, 以及它在哈佛商学院的作用。

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			<td height="40" style="height:40px;width:265px;">human umbilical vein endothelial cell</td>
			<td style="width:147px;">Fudan IBS Cell Center</td>
			<td style="width:343px;">FDCC-HXN180</td>
			<td style="width:761px;"></td>
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			<td height="40" style="height:40px;">dulbecco&#8217;s modified eagle&#8217;s medium&#160;</td>
			<td>Gibco</td>
			<td>11995040</td>
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			<td height="40" style="height:40px;">fetal bovine serum</td>
			<td>Gibco</td>
			<td>&#160;26400044</td>
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			<td height="40" style="height:40px;">PLKO.1-puro vector</td>
			<td>Addgene</td>
			<td>#8453</td>
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			<td height="40" style="height:40px;">packing plasmid psPAX2&#160;</td>
			<td>Addgene</td>
			<td>#12260</td>
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			<td>Addgene</td>
			<td>#12259</td>
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			<td height="40" style="height:40px;">3D round-bottom 96-well plates</td>
			<td>S-Bio</td>
			<td>MS-9096M</td>
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			<td height="40" style="height:40px;">matrigel</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td style="width:761px;"></td>
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			<td height="40" style="height:40px;">Opti-MEM medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td style="width:761px;">reduced serum medium&#160;</td>
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			<td height="40" style="height:40px;">15-well plate</td>
			<td>Ibidi</td>
			<td>81501</td>
			<td>Air bubbles in the gel can be reduced by equilibrating the &#956;&#8211;Slide angiogenesis before usage inside the incubator overnight</td>
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			<td height="40" style="height:40px;">endothelial cell growth supplements</td>
			<td>Sciencell</td>
			<td>#1052</td>
			<td style="width:761px;"></td>
		</tr>
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			<td height="40" style="height:40px;">10-cm culture dish</td>
			<td>Corning</td>
			<td>Scipu000813</td>
			<td></td>
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			<td height="40" style="height:40px;">Puromycin</td>
			<td>Gibco</td>
			<td>A1113802</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">typsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Automated Cell Counter System &#160;</td>
			<td>BioTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Image Analysis software&#160;</td>
			<td>Winmasis</td>
			<td style="width:343px;"></td>
			<td>http://mywim.wimasis.com&#160;</td>
		</tr>
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a comprehensive procedure to evaluate the in vitro performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay

冯·希佩尔·林道-林道 (VHL) 抑癌基因的失活在人类中枢神经系统 (CNS) 的小脑 (HBs) 的发展中起着至关重要的作用。然而, 哈佛商学院 (包括新生血管) 的细胞学起源和进化过程仍然存在争议, VHL-哈佛商学院的抗血管生成是基于典型的 HB 血管生成, 在临床试验中产生了令人失望的结果。抗血管治疗成功的临床翻译的一个主要障碍是对血管肿瘤的新生血管缺乏透彻的了解。在本文中, 我们提出了一个综合的程序来评价在体外是否存在经典的肿瘤血管生成在哈佛商学院, 以及它在哈佛商学院的作用。通过这一程序, 研究人员可以准确地了解乙肝新生血管的复杂性, 并确定其在哈佛商学院的这种常见的血管生成功能。这些协议可用于评估最有前景的肿瘤抗血管治疗, 它具有很高的翻译潜力, 无论是肿瘤治疗或协助优化抗血管生成治疗的哈佛商学院在未来的翻译。结果突出了血红蛋白新生血管的复杂性, 认为这种共同的形态血管生成只是血红蛋白新生血管的补充机制。

原图像采用倒置光镜。控件组和 VHL 组的典型图像显示在图 2A1和图 2A2中。对照组的发芽长度短于 VHL 组。
上传图像后, 在线平台直接提供分析结果。输出由两部分组成: 处理后的图像和相应的分析结果。控制组和 VHL 基因静默组的处理图像标记为不同的颜色 ...

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay at JoVE.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

本文提出了一个综合的方法来评价在体外是否存在经典的肿瘤血管生成在小脑 (哈佛商学院) 及其在哈佛商学院的作用。研究结果突出了血红蛋白-新生血管的复杂性, 认为这种共同的血管生成形式仅仅是血红蛋白-新生血管的补充机制。

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

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Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

In Vitro Assay to Study Tumor-macrophage Interaction

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...