舌细胞外基质的制备与舌鳞癌体外重建
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 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。 冷冻解冻细?…
Representative Results
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?…
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| C57-BL/6J mice | Sun Yat-sen University Laboratory Animal Center | ||
| Ethanol | Guangzhou Chemical Reagent Factory | HB15-GR-2.5L | |
| Sodium chloride | Sangon Biotech | A501218 | |
| Potassium chloride | Sangon Biotech | A100395 | |
| Dibasic Sodium Phosphate | Guangzhou Chemical Reagent Factory | BE14-GR-500G | |
| Potassium Phosphate Monobasic | Sangon Biotech | A501211 | |
| 1.5 mL EP tube | Axygen | MCT-150-A | |
| Ultra-low temperature freezer | Thermo Fisher Scientific | ||
| 3.5 cm cell culture dish | Thermo Fisher Scientific | 153066 | |
| 6 cm cell culture dish | Greiner | 628160 | |
| Triton X-100 | Sigma-Aldrich | V900502 | |
| Calcium chloride | Sigma-Aldrich | 746495 | |
| Magnesium chloride | Sigma-Aldrich | 449164 | |
| DNase | Sigma-Aldrich | D5025 | |
| Magnesium sulphate | Sangon Biotech | A601988 | |
| Glucose | Sigma-Aldrich | 158968 | |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | |
| Ampicillin | Sigma-Aldrich | A9393 | |
| Kanamycin | Sigma-Aldrich | PHR1487 | |
| Surgical suture | Shanghai Jinhuan | ||
| 250 mL wide-mouth bottle | SHUNIU | 1407 | |
| Magnetic stirrer | AS ONE | 1-4602-32 | |
| CO2 incubator | SHEL LAB | SCO5A | |
| 10 mL syringe | Hunan Pingan | ||
| 50 mL centrifuge tube | Greiner | 227270 | |
| Cal27 cell | Chinese Academy of Science, Shanghai Cell Bank | Tongue squamous cell carcinoma cell line | |
| U2OS cell | Chinese Academy of Science, Shanghai Cell Bank | Human osteosarcoma cell line | |
| DMEM/F12 | Sigma-Aldrich | D0547 | |
| Sodium pyruvate | Sigma-Aldrich | P5280 | |
| Hepes free acid | BBI | A600264 | |
| FBS | Hyclone | SH30084.03 | |
| 4 °C fridge | Haier | ||
| Water purifier | ELGA | ||
| Hemocytometer | BLAU | 717805 |
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Diesen Artikel zitieren
Yao, Y., Lin, W., Zhang, Y. Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro. J. Vis. Exp. (136), e57235, doi:10.3791/57235 (2018).