体外 富含透明质酸的细胞外基质对神经嵴细胞迁移的影响研究
Summary
该协议概述了适用于参与神经嵴细胞体内迁移到富含透明质的细胞外基质的分子的功能分析的体外迁移实验。
Abstract
神经嵴细胞（NCC）是起源于神经管背侧区域的高度迁移细胞。NCC从神经管迁移是NCC产生及其随后向靶位点迁移的重要过程。NCC的迁移途径，包括周围的神经管组织，涉及富含透明质酸（HA）的细胞外基质。为了模拟NCC从神经管迁移到这些富含HA的周围组织中，本研究建立了由HA（平均分子量：1，200-1，400 kDa）和I型胶原蛋白（Col1）组成的混合底物迁移测定。该迁移测定表明NCC细胞系O9-1细胞在混合基质上高度迁移，并且在迁移过程中HA涂层在粘附部位降解。该 体外 模型可用于进一步探索NCC迁移所涉及的机制基础。该协议也适用于评估不同的底物作为支架来研究NCC迁移。
Introduction
神经嵴细胞（NCC）是存在于发育中的胚胎中的多能细胞群，它们起源于神经形成过程中的神经板边界。它们有助于形成各种组织，包括周围神经系统、心血管系统、颅面组织和骨骼1。在神经板边界处诱导和NCC规范后，NCC从神经上皮迁移并迁移到NCC来源的组织部位1。
透明质酸（HA）是一种非硫酸化糖胺聚糖，作为细胞外基质（ECM）的组分分布在各种组织中。HA在胚胎发育中的重要性已在模型系统中通过消融负责透明质酸代谢的基因得到证实。例如，发现非洲爪蟾中透明质酸合酶基因（Has1和Has2）的突变会导致NCC迁移缺陷和颅面畸形2。此外，据报道，HA结合蛋白聚糖，聚集聚糖和保全糖对NCC迁移发挥抑制作用3。在小鼠中，Has2消融导致心内膜垫形成的严重缺陷，导致妊娠中期（E9.5-10）致死率4，5，6。
跨膜蛋白 2 （Tmem2） 是一种细胞表面透明质酸酶，最近已被证明通过去除粘附位点7，8 处的基质相关 HA，在促进整合素介导的癌细胞粘附和迁移中发挥关键作用。最近，Inubushi等人9证明，由于NCC迁移/迁移和存活的异常，Tmem2的缺乏会导致严重的颅面缺陷。在先前的研究9中，分析了NCC形成和迁移过程中Tmem2的表达。在NCC分层部位和迁移的Sox9阳性NCC中观察到Tmem2表达（图1）。此外，使用Tmem2耗尽的小鼠O9-1神经嵴细胞，研究表明Tmem2的体外表达对于O9-1细胞形成粘附并迁移到含HA的底物中至关重要（图2和图3）9。
这些结果强烈表明，Tmem2对于NCC通过富含HA的ECM的粘附和迁移也很重要。然而，富含HA的ECM中NCC粘附和迁移的分子机制尚不清楚。因此，有必要建立一个 体外 培养实验系统，以充分探索富含HA的ECM中的NCC粘附和迁移。
在测试细胞迁移的众多方法中，基于细胞伤口闭合的测定是生理学和肿瘤学领域经常使用的简单方法10。这种方法是有用的，因为它与 体内 表型相关，并且有效地确定药物和化学引诱剂在细胞迁移中的作用11。可以通过测量随时间变化的细胞间隙距离来评估整个细胞质量和单个细胞的迁移能力11。在本手稿中，介绍了 一种改良的 基于体外伤口闭合的测定法，以模拟NCC迁移到神经管周围富含HA的组织中。该程序也适用于研究不同的ECM成分（即胶原蛋白，纤连蛋白和层粘连蛋白），以分析ECM支架在NCC迁移中的作用。
Protocol
Representative Results
Discussion
各种 ECM 组件规范 NCC 迁移/迁移。例如，HA积极调节NCC迁移2，15。有趣的是，一项基于细胞表面透明质酸酶Tmem2的遗传小鼠模型的研究阐明了NCC迁移中HA降解的要求9。神经管周围的ECM中也含有丰富的胶原蛋白16。Decorin是一种富含亮氨酸的小蛋白聚糖，已被证明可以在神经发育过程中调节NCC迁移17。其他…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我非常感谢入江文俊和山口优在建立这种方法时给予的鼓励和善意的建议。这项工作得到了日本科学促进会（#19KK0232至T.I.，#20H03896至T.I.）的科学研究项目资助。Yamamoto等人（2017）8描述了在玻璃基板上涂覆HA和在基板上原位HA降解测定的原始方法，而Irie等人（2021）7描述了HA / Col1混合基板的制备方法。
Materials
| 10cm cell culture dish | CORNING | Cat. 353003 | |
| 1X PBS | Millipore | Cat. No. BSS-1005-B | |
| 2-well culture inserts | ibidi | Cat. No. 80209 | |
| Alexa 555-labelled goat anti-mouse IgG | Invitrogen | Cat. A21422 | Goat derived anti-mouse secondary antibody |
| automated cell counter | Bio-Rad | Cat. No. TC20 | |
| CELLBANKER | ZENOGEN PHARMA | Cat. 11910 | Cell freezing medium |
| collagen type I | Sigma | Cat. No. 08-115 | |
| Complete ES Cell Medium | Millipore | Cat. No. ES-101-B | |
| DAPI | Invitrogen | Cat. 10184322 | |
| Dulbecco’s Modified Eagle Medium | Gibco | Cat. 11971025 | |
| Fetal Bovine serum | Gibco | Cat. 10270106 | |
| fluorescence microscope | Keyence | Cat. No. BZ-X700 | |
| Fluoresent labelled HA | PG Research | FAHA-H2 | |
| Glas bottom dish | Iwaki | Cat. 11-0602 | |
| glutaldehyde | Sigma | Cat. No. G5882 | |
| Matrigel | Fisher | Cat. No. CB-40234 | The basement-membrane matrix |
| monoclonal anti-vinculin antibody | Sigma | Cat. No. V9264 | |
| mounting media | Dako | S3023 | |
| Normal goat serum | Fisher | Cat. 50062Z | |
| O9-1 cells | Millipore | Cat. No. SCC049 | |
| Paraformaldehyde | Sigma | Cat. 158127 | |
| triethoxysilane | Sigma | Cat. No. 390143 | |
| trypsin-EDTA | Millipore | Cat. No. SM-2003-C |
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