一种优化的O9-1/水凝胶系统，用于研究神经嵴细胞中的机械信号
Summary
这里描述了详细的分步方案，用于使用多能O9-1神经嵴细胞和不同硬度的聚丙烯酰胺水凝胶 在体外 研究机械信号。
Abstract
神经嵴细胞（NCCs）是脊椎动物胚胎多能细胞，可以迁移和分化成各种细胞类型，从而产生各种器官和组织。组织刚度产生机械力，这是一种在NCC分化中起关键作用的物理线索;然而，机制仍不清楚。这里描述的方法为优化生成具有不同刚度的聚丙烯酰胺水凝胶，精确测量这种刚度以及评估O9-1细胞中机械信号的影响提供了详细信息，O9-1细胞是一种模拟 体内 NCC的NCC系。
使用原子力显微镜（AFM）测量水凝胶刚度，并相应地指示不同的刚度水平。在不同硬度的水凝胶上培养的O9-1 NCCs表现出不同的细胞形态和应激纤维的基因表达，这表明机械信号变化引起的生物学效应不同。此外，这确定了改变水凝胶刚度导致有效的 体外 系统，通过改变凝胶刚度和分析NCC中的分子和遗传调节来操纵机械信号传导.O9-1 NCCs可以在相应分化介质的影响下分化成广泛的细胞类型，并且便于 在体外操纵化学信号。因此，这种 体外 系统是研究机械信号传导在NCC中的作用及其与化学信号相互作用的有力工具，这将有助于研究人员更好地了解神经嵴发育和疾病的分子和遗传机制。
Introduction
神经嵴细胞（NCCs）是脊椎动物胚胎发生过程中的一组干细胞，具有显着的迁移能力，有助于各种器官和组织的发育。NCCs可以分化成不同的细胞类型，包括感觉神经元，软骨，骨骼，黑素细胞和平滑肌细胞，这取决于轴向起源的位置和NCC1，2的局部环境引导。由于能够分化成多种细胞类型，在神经嵴（NC）发育的任何阶段引起失调的遗传异常都可能导致许多先天性疾病2。例如，NCC形成，迁移和发展过程中的扰动导致发育障碍，统称为神经性心脏病1，3。这些疾病的范围从由于NCC形成失败而导致的颅面缺陷，例如Treacher Collins综合征，到由于NCC转移性迁移能力引起的各种癌症的发展，如黑色素瘤3，4，5，6所示。在过去的几十年里，研究人员对NCC在发育和疾病中的作用和机制有了非凡的发现，大多数发现都集中在化学信号7，8上。最近，机械信号已被证明在NCC开发9，10中发挥了关键但知之甚少的作用。
NCC的环境线索在其发育过程中起着至关重要的作用，包括调节NCC分化成各种细胞类型。环境线索，例如物理线索，影响关键行为和细胞反应，如功能多样化。机械转导允许细胞感知并响应这些线索，以维持各种生物过程2。NCCs被邻近细胞和不同的底物包围，例如细胞外基质（ECM），其可以产生机械刺激以维持体内平衡并通过命运决定，增殖和凋亡来适应变化11。机械转导从发生机械细胞外刺激的感觉成分的质膜开始，导致细胞12的细胞内调节。整合素、局灶粘附和质膜的连接将机械信号（如剪切力、应力和周围基质的刚度）传递到化学信号中，以产生细胞响应12。化学信号从质膜到最终细胞调节的传递是通过不同的信号通路进行的，以最终确定生物体的重要过程，例如分化。
一些研究表明，来自底物刚度的机械信号在细胞分化中起作用13，14。例如，先前的研究表明，在软底物上生长的间充质干细胞（MSCs）具有类似于脑组织的刚度（在0.1-1.0 kPa的范围内）导致神经元细胞分化15，16。然而，当在模仿肌肉硬度的8-17 kPa底物上生长时，更多的MSCs分化成肌细胞样细胞，而当MSCs在坚硬的底物（25-40 kPa）上培养时观察到成骨细胞样分化15，16。机械信号通路中的不规则和异常突出了机甲转导的重要性，这些异常和异常可能导致严重的发育缺陷和疾病，包括癌症，心血管疾病和骨质疏松症17，18，19。在癌症中，正常的乳房组织是柔软的，并且在僵硬和致密的乳房组织中患乳腺癌的风险增加，这种环境更类似于乳腺肿瘤15。有了这些知识，可以通过体外系统对底物刚度的简单操作来研究机械信号传导对NCC发展的影响，为理解NC相关疾病进展和病因的基本原理提供了进一步的优势和可能性。
为了研究机械信号对NCC的影响，我们基于先前发表的方法的优化和对NCC对不同机械信号的响应的评估，建立了一个有效的NCC体外系统20，21。为改变水凝胶刚度制备和评估机械信号传导在NCC中的影响提供了详细的方案。为了实现这一点，使用O9-1 NCCs作为NC模型来研究对刚性水凝胶与软水凝胶的反应和变化。O9-1 NCCs是在第8.5天从小鼠胚胎（E）中分离出来的稳定NC细胞系。O9-1 NCCs在体内模仿NCC，因为它们可以在定义的分化培养基22中分化成各种NC衍生的细胞类型。为了研究NCC的机械信号传导，从不同浓度的丙烯酰胺和双丙烯酰胺溶液中制备具有可调弹性的基质基质，以达到所需的刚度，与生物底物刚度20，21，23相关。为了优化NCC，特别是O9-1细胞的基质底物的条件，对先前发表的方案20进行了修改。该协议中的一项更改是在37°C下将水凝胶在胶原I中孵育，用0.2%乙酸稀释而不是50mM HEPES过夜。乙酸的低pH值导致均匀分布和更高的胶原I掺入，从而允许ECM蛋白24更均匀地附着。此外，在将水凝胶储存在培养箱中之前，分别在磷酸盐缓冲盐水（PBS）中以10%和5%的浓度使用马血清和胎牛血清（FBS）的组合。马血清被用作FBS的额外补充，因为它能够在10%25的浓度下促进细胞增殖和分化。
使用这种方法，通过ECM蛋白质包衣（例如胶原蛋白I）模拟生物环境，为NCC的生长和存活创造准确的体外环境20，21。通过原子力显微镜（AFM）定量分析所制备的水凝胶的刚度，原子力显微镜是描述弹性模量26的众所周知的技术。为了研究不同硬度水平对NCC的影响，在水凝胶上培养和制备野生型O9-1细胞，用于免疫荧光（IF）染色，以对抗丝状肌动蛋白（F-actin），以显示细胞粘附和形态的差异响应底物刚度的变化。利用这种体外系统，研究人员将能够研究机械信号在NCC中的作用及其与其他化学信号的相互作用，以更深入地了解NCC与机械信号之间的关系。
Protocol
Representative Results
Discussion
本研究的目标是提供一种有效和高效的 体外 系统，以更好地了解机械信号对NCC的影响。除了遵循上述分步方案外，研究人员还需要记住，O9-1 NCC的细胞培养受到用于制备水凝胶的玻璃盖玻片类型的影响。例如，有人指出，接种在特定类型的玻璃盖玻片上的细胞（见 材料表）存活并增殖良好，而接种在其他类型的玻璃盖玻片上的培养细胞显示出更多的细胞死亡。此外，严格遵循…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢德克萨斯大学健康科学中心原子力显微镜-UT核心设施的操作员Ana-Maria Zaske博士在该项目中为AFM提供的专业知识。我们还感谢美国国立卫生研究院的资金来源（K01DE026561，R03DE025873，R01DE029014，R56HL142704和R01HL142704给J. Wang）。
Materials
| 12 mm #1 Corning 0211 Glass Coverslip | Chemglass Life Sciences | CLS-1763-012 | |
| 2% Bis-Acrylamide | Sigma Aldrich | M1533 | |
| 24-well plate | Greiner Bio-one | 662165 | |
| 25 mm #1 Corning 0211 Glass Coverslip | Chemglass Life Sciences | CLS-1763-025 | |
| 3-aminopropyl triethoxysilane (APTS) | Sigma Aldrich | A3648 | |
| 4-well cell culture plate | Thermo Scientific | 179830 | |
| 4% Paraformaldehyde | Sigma Aldrich | J61899-AP | |
| 40% Acrylamide | Sigma Aldrich | A4058 | |
| 50% glutaraldehyde | Sigma Aldrich | G7651 | |
| 6-well cell culture plate | Greiner Bio-one | 657160 | |
| AFM cantilever (spherical bead) | Novascan | ||
| AFM software | Catalyst NanoScope | Model: 8.15 SR3R1 | |
| Alexa Fluor 488 Phalloidin | Thermo Fisher | A12379 | |
| Ammonium Persulfate (APS) | Sigma Aldrich | 248614 | Powder |
| anti-AP-2α Antibody | Santa Cruz | sc-12726 | |
| anti-Vinculin antibody | Abcam | ab129002 | |
| Atomic Force Microscopy (AFM) Bioscope Catalyst | Bruker Corporation | ||
| Collagen type I (100mg) | Corning | 354236 | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher | D1306 | |
| Dichloromethylsilane (DCMS) | Sigma Aldrich | 440272 | |
| Donkey serum | Sigma Aldrich | D9663 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Corning | 10-017-CV | |
| Fetal bovine serum (FBS) | Corning | 35-010-CV | |
| Fluorescence microscope | Leica | Model DMi8 | |
| Fluoromount-G mounting medium | SouthernBiotech | 0100-35 | |
| HEPES | Sigma Aldrich | H3375 | Powder |
| Horse serum | Corning | 35-030-CI | |
| iScript Reverse Transcription Supermix | Bio-Rad | 1708841 | |
| Penicillin-Streptomycin antibiotic | Thermo Fisher | 15140148 | |
| RNeasy micro kit | Qiagen | 74004 | |
| Sterile 1x PBS | Hyclone | SH30256.02 | |
| Sterile deionized water | Hardy Diagnostics | U284 | |
| sulfo-SANPAH | Thermo Fisher | 22589 | |
| SYBR green | Applied Biosystems | 4472908 | |
| TEMED | Sigma Aldrich | T9281 | |
| Triton X-100 | Sigma Aldrich | X100 | |
| Tween 20 | Sigma Aldrich | P9416 |
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