人瘢痕疙瘩组织中原代真皮成纤维细胞的分离、培养和表征
Summary
本研究描述了一种优化的方案，用于从瘢痕疙瘩组织中建立原代成纤维细胞，可以有效和稳定地提供纯净和可行的成纤维细胞。
Abstract
成纤维细胞是瘢痕疙瘩组织中的主要细胞类型，在瘢痕疙瘩的形成和发展中起着至关重要的作用。来自瘢痕疙瘩组织的原代成纤维细胞的分离和培养是进一步研究瘢痕疙瘩的生物学功能和分子机制以及治疗瘢痕疙瘩的新治疗策略的基础。获得原代成纤维细胞的传统方法具有局限性，例如细胞状态差，与其他类型的细胞混合以及易受污染。本文描述了一种优化且易于重现的方案，可以减少获取成纤维细胞时可能出现问题的发生。在该方案中，可以在分离后5天观察到成纤维细胞，并在培养10天后达到近80%的汇合度。然后，使用用于免疫荧光测定的PDGFRα和vimentin抗体以及用于流式细胞术的CD90抗体传代和验证成纤维细胞。总之，通过该方案可以轻松获得来自瘢痕疙瘩组织的成纤维细胞，这可以为瘢痕疙瘩研究提供实验室中丰富而稳定的细胞来源。
Introduction
瘢痕疙瘩是一种纤维增殖性疾病，表现为斑块的持续生长，这些斑块经常侵入周围的正常皮肤而没有自限性，并引起患者不同程度的瘙痒、疼痛以及美容和心理负担1.成纤维细胞是参与瘢痕疙瘩的原代细胞，通过过度增殖、多余的细胞外基质产生和无序的胶原蛋白在这种疾病的形成和发展中起着至关重要的作用2,3。然而，潜在的发病机制尚不清楚，仍然缺乏有效的瘢痕疙瘩治疗方法;因此，迫切需要进一步研究4,5。
由于体内瘢痕疙瘩研究没有理想的动物模型6,7，通过从瘢痕疙瘩组织中获取原代成纤维细胞来构建体外模型可以为瘢痕疙瘩研究提供可行性和可靠性2,6。原代细胞是直接来源于活组织的细胞，人们普遍认为，与细胞系8,9相比，这些细胞可以更接近多个个体的生理状态和遗传背景。原代细胞培养为研究细胞的生长和代谢以及其他细胞表型提供了一种强有力的手段。
目前，获得原代成纤维细胞的方法有两种：酶消化和外植体培养。然而，已经确定了获得原代成纤维细胞的几个障碍，例如被各种细菌或真菌污染的风险，与不易去除的其他类型的细胞混合，培养周期的长周期，与原始细胞相比细胞特性的后续变化，等等9.因此，开发一种可行且有效的方法来获得原代成纤维细胞是进一步研究和应用的基础。本研究描述了一种优化的方案，用于从瘢痕疙瘩组织中提取原代成纤维细胞，可以有效和稳定地提供纯净和可行的成纤维细胞。
Protocol
这项研究得到了南方医科大学皮肤病医院机构审查委员会的批准（2020081）。在从个体收集组织之前，已获得知情的患者同意。 1. 准备 注意：以下程序应在生物安全柜下的无菌环境中进行。 通过将 10% 胎牛血清 （FBS） 和 1% 青霉素-链霉素-两性霉素 B 溶液 （PSA） 添加到高葡萄糖 Dulbecco 的改良 Eagle 培养基 （DMEM） 中来制备完整的培养?…
Representative Results
该协议的时间线总结在 图1A中。分离过程的一些代表性图像如图 2所示;小心地去除表皮和脂肪层，并将真皮层分离成3-4mm2的小碎片，接种到培养皿中。 如图3A所示，在处理后5天在显微镜下观察到组织碎片的几个成纤维细胞生长。如图3B所示，成纤维细胞显示出高增殖速率，并…
Discussion
从瘢痕疙瘩组织获得原代成纤维细胞是进一步研究的关键基础。到目前为止，有两种方法可以获得原代成纤维细胞：酶消化和外植体培养11,12,13,14。然而，两种传统方法都有局限性，例如易受污染，与其他类型的细胞混合，培养期长，成功率低15,16。这项研?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金（批准号81903189和82073418）和广州市科学技术基金（批准号202102020025）的资助。
Materials
| 1.5 mL sterile centrifuge tube | JETBIOFIL | CFT002015 | |
| 15 mL sterile centrifuge tube | JETBIOFIL | 8076 | |
| 4% polyformaldehyde | Beyotime Biotechnology | P0099 | Cell fixation |
| 50 mL sterile centrifuge tube | JETBIOFIL | 8081 | Put keloid tissue |
| Alexa Fluor-555 goat anti-rabbit IgG | Abcam | Alexa Fluor 555 | second antibody for immunofluorescence staining assay |
| Anti human CD90 | BioLegend | B301002 | Identify the purity of fibroblasts |
| Antibody diluent | Beyotime Biotechnology | P0262 | |
| Biological safety cabinet | Thermo Scientific | 1300 series A2 | Isolation and culture cells |
| Bovine serum albumin | aladdin | B265993 | Blocking for immunofluorescence staining assay |
| Carbon dioxide incubator | ESCO | CCL-170B-8 | Using for culturing cells |
| Cell cryotubes | Corning | 43513 | Store the cells in low temperature |
| centrifugal machine | Thermo Fisher | ST 16R | Discard supernatant |
| DAPI | Beyotime Biotechnology | C1006 | Stain the cellular nucleus |
| DMSO | MP Biomedicals | 196055 | Using for preserving cells |
| Dulbecco's modified eagle medium | Gibco | C11995500BT | Culture medium solution |
| Fetal bovine serum | BI | 04-001-1A | |
| Flow cytometer | BD | BD FACSCelesta | Observing the identity of cells |
| frozen box | Thermo Scientific | 5100-0050 | |
| Inverted microscope | Nikon | ECLIPSE Ts2 | |
| Laser confocal microscope | Nikon | AIR-HD25 | Observing the immunofluorescence staining assay |
| PDGFR-α antibody | CST | 3174T | First antibody for immunofluorescence staining assay |
| Penicillin-streptomycin-Am solution | Solarbio | P1410 | Add in culture medium solution to avoid contamination |
| petri dish | JETBIOFIL | 7556 | Culture fibroblasts |
| Phosphate buffered saline solution | Gibco | C10010500BT | Culture medium solution |
| Rabbit (DAIE) mAB IgG XR (R) Isotuge Control (PE) | Cell Signaling Technology | 5742S | As a control for flow cytometry |
| Round coverslip | Biosharp | 801007 | Cell culture |
| Triton X 100 | Solarbio | T8200 | Punch holes in the cell membrane |
| Trypsin-EDTA | Gibco | 25200072 | Used for passaging cells |
| Vimentin antibody | Abcam | ab8978 | First antibody for immunofluorescence staining assay |
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Citar este artigo
Song, J., Zhang, Y., Pan, H., Xu, X., Deng, C., Yang, B. Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue. J. Vis. Exp. (197), e65153, doi:10.3791/65153 (2023).