Name: fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro
Uploaded: 2018-06-20T12:30:00
Description: Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

作者对国家自然科学基金 (31371390)、国家高新技术发展项目 (2014AA020702) 和广东省科技计划 (2016B030231001) 的研究资助给予了认可。

所有动物工作都是按照《动物福利法》、《机构指南》和中山大学动物保育和使用委员会批准的。
1. 制备 TEM
<ol><li>用不育的手术剪刀和镊子来执行小鼠颈椎脱位并去除舌头。</li>
	<li>浸泡在75% 乙醇3分钟的舌头, 然后把每一个舌头到1.5 毫升离心 (EP) 管与1毫升的10毫米无菌磷酸盐缓冲溶液 (PBS)。 注意: PBS 在以下所有步骤中的浓度与此步骤中的浓度相同。</li>
	<li>冷冻解冻细?...

<ol>
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Oncol. 29, 1488-1494 (2011).</li><li>Zhao, L., Huang, L., Yu, S., Zheng, J., Wang, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+tongue+tissue+as+an+in+vitro.+model+for+studying+tongue+cancer+and+tongue+regeneration.">Decellularized tongue tissue as an in vitro. model for studying tongue cancer and tongue regeneration.</a> Acta Biomaterialia. 58, 122-135 (2017).</li><li>Ng, S. L., Narayanan, K., Gao, S., Wan, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restricted+progenitors+for+the+repopulation+of+decellularized+heart.">Lineage restricted progenitors for the repopulation of decellularized heart.</a> Biomaterials. 32, 7571-7580 (2011).</li><li>Ott, H. 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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=Initial+binding+and+recellularization+of+decellularized+mouse+lung+scaffolds+with+bone+marrow-derived+mesenchymal+stromal+cells.">Initial binding and recellularization of decellularized mouse lung scaffolds with bone marrow-derived mesenchymal stromal cells.</a> Tissue Eng. Pt A. 18, 1-16 (2012).</li><li>Ott, H. 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=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat. Med. 16, 927-933 (2010).</li><li>Petersen, T. 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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=Comparative+assessment+of+detergent-based+protocols+for+mouse+lung+de-cellularization+and+re-cellularization.">Comparative assessment of detergent-based protocols for mouse lung de-cellularization and re-cellularization.</a> Tissue Eng. Part C-ME. 18, 420-432 (2012).</li><li>Ross, E. 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=Embryonic+stem+cells+proliferate+and+differentiate+when+seeded+into+kidney+scaffolds.">Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds.</a> J. Am. Soc. Nephrol. 20, 2338-2347 (2009).</li><li>Song, J. J., Guyette, J. P., Gilpin, S., Gonzalez, G., Vacanti, J. P., Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney.">Regeneration and experimental orthotopic transplantation of a bioengineered kidney.</a> Nat. Med. 19, 646-651 (2013).</li><li>Sullivan, D. 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=Decellularization+methods+of+porcine+kidneys+for+whole+organ+engineering+using+a+high-throughput+system.">Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system.</a> Biomaterials. 33, 7756-7764 (2012).</li><li>Soto-Gutierrez, A., Wertheim, J. A., Ott, H. C., Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+whole-organ+assembly:+moving+toward+transplantation+on+demand.">Perspectives on whole-organ assembly: moving toward transplantation on demand.</a> J. Clin. Invest. 122, 3817-3823 (2012).</li><li>Song, J. J., Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+engineering+based+on+decellularized+matrix+scaffolds.">Organ engineering based on decellularized matrix scaffolds.</a> Trends Mol. Med. 17, 424-432 (2011).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-organ+tissue+engineering:+decellularization+and+recellularization+of+three-dimensional+matrix+scaffolds.">Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds.</a> Annu. Rev. Biomed. Eng. 13, 27-53 (2011).</li><li>Shamis, Y., 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=Organ-specific+scaffolds+for+in+vitro+expansion,+differentiation,+and+organization+of+primary+lung+cells.">Organ-specific scaffolds for in vitro expansion, differentiation, and organization of primary lung cells.</a> Tissue Eng. Part C-ME. 17, 861-870 (2011).</li><li>Nakayama, K. H., Batchelder, C. A., Lee, C. I., Tarantal, A. 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一个完善的瓣膜 ecm 制造协议应保留本机 ecm 成分, 同时去除组织中的细胞成分几乎完全21。尽管目前报告的去细胞协议需要通过血管灌流来去除蜂窝材料通过对流运输, 这里采用了机械搅拌, 称为传统的简单和廉价的方法22,23,24,25,26. 此外, 由于舌头富含 lingual...

舌有多种重要功能, 如吞咽、发音和味觉。因此, 舌功能的损害对患者的生活质量有很大的影响1。口腔内最常见的恶性肿瘤是舌鳞状细胞癌 (TSCC), 通常发生在饮酒或吸烟2的人身上。
近年来, TSCC 的基础研究取得了很小的进展。缺乏有效的体外研究模式仍然是最大的问题之一。因此, 细胞外基质 (ECM) 结果是一个潜在的解决方案。由于 ecm 是由高度组织的矩阵元件组成的复杂网络框架, 因此具有 ecm 类结构和成分的脚手架材料将胜任癌症研究。瓣膜 ecm 能够完美地为细胞从同一来源的体外提供利基, 这是 ecm 最显著的优势。
ECM 可以通过去细胞使用洗涤剂和酶将细胞成分从组织中去除。各种 ECM 组件, 包括胶原蛋白、纤连蛋白和瓣膜基质层粘连蛋白, 为培养细胞提供了一种类似于组织的微环境, 促进了细胞的存活、增殖和分化3。此外, 在 ECM 中缺乏细胞成分的情况下, 移植的免疫原性可降至最低水平。
到目前为止, 瓣膜 ECM 的制备方法已经尝试了不同的组织和器官, 如心脏4,5,6,7, 肝8,9,10 ,11, 肺12,13,14,15,16,17, 和肾脏18,19,20. 然而, 在我们最了解的情况下, 没有就舌头上的类似工作找到相关的研究。
在本研究中, 瓣膜舌外基质 (TEM) 是通过一系列物理、化学和酶处理, 高效、廉价地制备出来的。然后用透射电镜对体外TSCC 进行了重述, 对 TSCC 的行为和发育进行了适当的模拟。tem 具有良好的生物相容性以及引导细胞到组织特异性的能力, 这表明 tem 在 TSCC 研究3中可能具有很大的潜力。此处显示的协议为研究 TSCC 的发病机制或临床治疗的研究者提供了选择。

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			<td height="42" style="height:42px;width:216px;">C57-BL/6J mice</td>
			<td style="width:292px;">Sun Yat-sen University Laboratory Animal Center</td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">HB15-GR-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A100395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dibasic Sodium Phosphate</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">BE14-GR-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic&#160;</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL EP tube</td>
			<td style="width:292px;">Axygen</td>
			<td style="width:119px;">MCT-150-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-low temperature freezer&#160;</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3.5 cm cell culture dish</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">153066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 cm cell culture dish</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">V900502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">449164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium sulphate</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A601988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">158968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">PHR1487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical suture</td>
			<td style="width:292px;">Shanghai Jinhuan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 mL wide-mouth bottle</td>
			<td style="width:292px;">SHUNIU</td>
			<td style="width:119px;">1407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stirrer</td>
			<td style="width:292px;">AS ONE</td>
			<td style="width:119px;">1-4602-32</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">CO2 incubator</td>
			<td style="width:292px;">SHEL LAB</td>
			<td style="width:119px;">SCO5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL syringe</td>
			<td style="width:292px;">Hunan Pingan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL centrifuge tube</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">227270</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cal27 cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Tongue squamous cell carcinoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">U2OS cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes free acid</td>
			<td style="width:292px;">BBI</td>
			<td style="width:119px;">A600264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
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			<td style="width:119px;">SH30084.03</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4 &#176;C fridge</td>
			<td style="width:292px;">Haier</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water purifier</td>
			<td style="width:292px;">ELGA</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:292px;">BLAU</td>
			<td style="width:119px;">717805</td>
			<td></td>
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fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

为了构建一种有效的、逼真的舌鳞癌 (TSCC) 模型, 建立了瓣膜舌细胞外基质 (TEM), 为 TSCC 的构建提供了功能性支架。TEM 为细胞生长、分化和细胞迁移提供了体外小生境。本机细胞外基质 (ECM) 的显微结构和瓣膜基质中保留的生化成分为锚固细胞提供组织特异的龛位。经脱氧核糖核酸酶 (DNase) 消化, 同时具有严重的有机或无机预处理, 可实现透射电镜的制备。该协议易于操作, 并确保了去细胞的高效率。透射电镜显示, 在静态或搅拌培养条件下, TSCC 细胞具有良好的细胞相容性, 使 TSCC 模型得以构建。自制的生物反应器也用于细胞培养的持续搅拌条件。应用 TEM 重建 TSCC 显示了类似临床 TSCC 组织病理学的特点和性质, 提示 TSCC 研究的潜力。

本议定书的编写 TEM 证明是有效和适当的。与母语组织相比, 透射电镜显示出完美的去细胞。去细胞的疗效经苏木精-伊红染色 (图 1A B) 证实。he 染色结果显示透射电镜核染色完全消失 (图 1B)。此外, 从先前的工作中 dna 含量的量化表明, dna 几乎完全从 TEM3中去除。该协议在去除细胞成分?...

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

舌细胞外基质的制备与舌鳞癌体外重建

给出了一种有效去细胞的舌胞外基质 (TEM) 的制备方法。透射电镜可作为功能性支架, 用于在静态或搅拌培养条件下重建舌鳞癌 (TSCC) 模型。

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Research

JoVE Journal

Cancer Research

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

科研

癌症研究

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

研究使用细胞外基体水凝胶的正常组织辐射效应

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

M1巨噬细胞-4T1小鼠乳腺癌细胞在共培养中噬菌体活性的时移2D成像

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...