利用大分子挤出的人类食性增大疤痕的体外模型
Summary
该协议描述了使用大分子挤拥创建体外人类肥肥疤痕组织模型，类似于在体内条件。在拥挤的大分子环境中培养时，人类皮肤成纤维细胞表现出类似疤痕组织的表型、生物化学、生理学和功能特性。
Abstract
研究表明，体内组织中蛋白质、核酸、核糖蛋白、多糖等非常拥挤。以下协议应用大分子挤拥 （MMC） 技术，通过在体外添加中性聚合物（挤出体）来模拟这种生理挤拥。以前使用Ficoll或dextran作为人群的研究表明，使用MMC技术，在WI38和WS-1细胞系中胶原蛋白I和纤维素的表达得到显著增强。然而，这种技术尚未在原发肥疤源人类皮肤成纤维细胞（hHSFs）中得到验证。由于胶原蛋白的过度沉积会产生肥大疤痕，该协议旨在通过将MMC技术与hHSF一起应用，构建富含胶原蛋白的体外增肥疤痕模型。与传统二维（2-D）细胞培养系统相比，这种优化的MMC模型与体内疤痕组织具有更多的相似性。此外，与动物模型相比，它具有成本效益、时间高效且合乎道德。因此，此处报告的优化模型为肥肥疤痕相关研究提供了先进的”体内类似”模型。
Introduction
疤痕组织代表组织修复的终点。然而，在许多个人，特别是那些遭受烧伤或创伤1，疤痕可能是过度的，并施加不良的影响，愈合的皮肤的形态和功能。虽然病理（肥大疤痕和酮）疤痕形成的确切机制尚未完全理解，但胶原蛋白在伤口愈合过程中的过度沉积已被证明是一个必不可少的贡献2。
众所周知，转化生长因子β 1 （TGF-+1） 和 α 平滑肌肉作用素 （#SMA） 在增肥疤痕的形成中起着关键作用。有证据表明，升高的TGF-β1通过调节SMAD信号通路3直接刺激胶原蛋白的过度沉积。此外，在伤口愈合过程中，通过调节细胞收缩和重皮化，发现αSMA有助于肥大疤痕的形成。缺乏适当的体外和体内模型是开发和评估疤痕补救干预措施和疗法的主要障碍。本研究的目的是利用现有的MMC技术，构建一个”体内类似”肥肥疤痕模型，适合评估新的和新兴的疤痕相关干预。
多年来，在科学界，在身体外复制活组织一直是一个目标。二十世纪初体外技术的发展部分实现了这一目标。目前的体外技术从Roux最初的证明中略有进化，即胚胎细胞可以在温暖的盐水5中存活几天。然而，体外方法大多限于在二维培养的单细胞类型，并且不能准确地重述体内的细胞组织。虽然可用于检查细胞生物化学、生理学和遗传学，但原生组织是三维组织，并包含多种细胞类型。简单的二维体外系统将哺乳动物细胞置于高度人工的环境中，其中原生组织特定的结构在6中丢失。反过来，这影响细胞内和细胞外事件，导致异常细胞形态，生理和行为7。
该协议背后的兴趣在于肥肥疤痕和酮类物的开发和临床管理。虽然众所周知，皮肤纤维细胞在很大程度上是导致疤痕组织中存在胶原蛋白的大量生产的原因，但使用二维体外系统培育皮肤纤维细胞不能重现体内8中观察到的胶原蛋白的周转。当代体外方法基本上仍然使用”暖盐水”，这种环境与活体组织中的环境完全不同。体内组织非常拥挤，蛋白质、核酸、核糖蛋白和多糖，占总体积的5%-40%。由于没有两个分子可以同时占据相同的空间，因此几乎没有可用的可用空间，而且几乎完全没有自由水9。
MMC 技术施加了影响细胞溶质和间质流体热力学特性的限制。分子相互作用、受体-配体信号复合物、酶和细胞器被限制和限制自由相互作用。近细胞环境中的相互作用（即间室）也受到限制。近期证据证实，高浓度惰性大分子在拥挤的溶液扰动扩散，物理关联，粘度和水动力特性10。
有趣的是，几种流行的挤水剂（即菲科尔、dextran、聚乙烯丙酮[PVP]和钠4苯乙烯硫化酸[PSS]）在应用于不同的细胞类型和不同设置时不等效。在以前的一项研究中，菲科尔与PVP相比，中质干细胞的细胞毒性较小。这些结果被解释为其中性电荷和相对较小的水动力半径11的结果。与此相反，第二项研究发现，与Ficoll12相比，dextran在刺激人肺成纤维细胞的胶原蛋白I沉积方面更有效。来自我们自身研究的数据表明，Ficoll通过肥大疤痕衍生成纤维细胞增强胶原蛋白沉积，而PVP对这些细胞13有毒。
实践证明，在高度拥挤的体内环境中，原胶原蛋白转化为胶原蛋白的速度更快，而生物反应的速度在稀释的二维培养系统15中延迟。我们在这里优化了体外方案，纳入了MMC，以表明皮肤纤维成纤维细胞的培养，作为皮肤纤维化和疤痕形成更”体内”的模型。与常见的二维培养系统相比，用MMC培养hHSF能显著刺激胶原蛋白的生物合成和沉积。值得注意的是，纤维化的其他特性（即，增加基质金属蛋白酶[MP]和亲炎细胞因子的表达）也在这个优化的MMC协议13下明显。使用这种方法培养时，结果表明，皮肤细胞重述了体内测量的生理、生化和功能参数。
优化的MMC体外方案已用于评估胶原蛋白和其他ECM蛋白的表达，通过从肥大疤痕真皮分离的皮肤成纤维细胞和未介入的相邻真皮。在体外MMC环境中培养时，观察到hHSFs具有某些特性（即mRNA、生物化学、生理学和表型），类似于体内皮肤增生性疤痕组织。证据表明，物理和化学特性是选择挤出体和优化MMC条件进行体外培养的重要考虑因素。
为了证明原理，MMC协议在这里应用，以定性和定量评估Shikonin及其模拟诱导凋亡的能力。这允许评估这些天然中药（TCM）化合物在管理皮肤疤痕方面的潜在应用。尽管如此，这项体外MMC协议的简单性、成本效益和及时性也满足了欧盟指令2010/63/EU和美国环境保护署（EPA）最近关于消除哺乳动物实验的规定。
Protocol
Representative Results
Discussion
该协议旨在优化和验证人类皮骨疤组织改进的”疤痕在一罐”体外模型。此前的研究曾报告将MMC技术应用于人类肺成纤维细胞12个，人类骨髓中皮干细胞23个，以及人类皮肤纤维细胞23个，使用dextran12、Ficoll12和PVP23作为23人群。在此报道的研究中，先前公布的肥大疤痕衍生人类皮肤成纤维细胞?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了新加坡科学、技术和研究机构”SPF 2013/004：皮肤生物学基础研究”和”热带病护理创新”IAF-PP/2017 （HBMS） H17/01/a0/009 的资助。作者感激地感谢保拉·本尼博士和迈克尔·拉古纳博士的建议和帮助。
Materials
| 0.2 μm filter | Sartorius | 16534 | |
| 2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
| 4’,6-diamidino-2-phenylindole (DAPI) | Thermo Fisher Scientific | P36962 | |
| Alexa Fluor 680 | Thermo Fisher Scientific | A-21076 | |
| Alexa Fluor 800 | Thermo Fisher Scientific | A-11371 | |
| alpha smooth muscle actin (αSMA) primary antibody | Abcam | ab5694 | |
| Applied Biosystems 7500 Fast Real-Time PCR System (thermal cycler ) | Thermo Fisher Scientific | 4351106 | |
| Ascorbic acid | Wako | #013-12061 | |
| Bovine serum albumin | Sigma-Aldrich | #A2153 | |
| Bradford protein assay | Bio-Rad | 500-0006 | |
| Collagen I primary antibody (for immunostaining) | Abcam | 6308 | |
| Collagen I primary antibody (for western blot) | Abcam | ab21286 | |
| Collagen III primary antibody | Abcam | ab7778 | |
| Collagen IV primary antibody | Abcam | ab6586 | |
| Direct Red 80 | Sigma-Aldrich | 2610108 | |
| Dulbecco's Modified Eagle's Medium (DMEM) | Life Technologies | 11996-065 | |
| Fetal calf serum (FCS) | Life Technologies | 6000-044 | |
| Ficoll 400 | GE HealthCare | #17-0300-10 | |
| Ficoll 70 | GE HealthCare | #17-0310-10 | |
| GAPDH primary antibody | Sigma-Aldrich | G8795 | |
| Goat Anti-Rabbit secondary antibody | Abcam | ab97050 | |
| Human hypertrophic scar/normal fibroblasts (hHSF/hNSF) | Cell Research Corporation | 106, 107, 108 | |
| iScript cDNA Synthesis Kit | Bio-Rad | #1708890 | |
| MMP-1 primary antibody | Abcam | ab38929 | |
| MMP-13 primary antibody | Abcam | ab39012 | |
| MMP-2 primary antibody | Abcam | ab37150 | |
| MMP-9 primary antibody | Abcam | ab38898 | |
| NanoDrop Microvolume Spectrophotometers | Thermo Fisher Scientific | N/A | |
| Nitrocellulose membrane | Bio-Rad | 10484060 | |
| NuPAGE 4-12% Bis-Tris Protein Gels | Thermo Fisher Scientific | NP0321BOX | |
| Odyssey blocking buffer | LI-COR Biosciences | 927–40000 | |
| Odyssey Fc Imaging System | LI-COR Biosciences | N/A | |
| Olympus IX-81 HCS microscope (for immunostaining) | Olympus | N/A | |
| Penicillin/streptomycin solution (P/S) | Life Technologies | 15140-122 | |
| PrimePCR Assays | Bio-Rad | Customized primers pre-coated in 96-well plates based on requirement | |
| Protease inhibitor cocktail (PIC) | Sigma-Aldrich | 11697498001 | |
| PVP 360 | Sigma | #PVP360 | |
| PVP 40 | Sigma | #PVP40 | |
| RIPA buffer | Merck | R0278 | |
| RNeasy Plus Mini Kit | QIAGEN | #74134 | |
| Sodium vanadate | Sigma-Aldrich | 450022 | |
| Sodium vanadate | Sigma-Aldrich | 450243 | |
| SpectraMax M5 Multi-Mode microplate reader | Molecular Devices | N/A | |
| SsoAdvanced universal SYBR green supermix | Bio-Rad | #172-5270 | |
| Tween 20 | Sigma-Aldrich | P9416 |
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