成人心脏纤维细胞和肌纤维细胞的分离与表征
Summary
获得成纤维细胞的纯种群对于研究其在伤口修复和纤维化中的作用至关重要。这里描述的是一种详细的方法，从未受伤和受伤的小鼠心脏中分离成纤维细胞和肌纤维细胞，然后通过免疫荧光、RTPCR、荧光辅助细胞分拣和胶原蛋白凝胶收缩。
Abstract
心脏纤维化对损伤的反应是伤口愈合的生理反应。已努力研究和靶向减轻纤维化的纤维细胞亚型。然而，由于缺乏普遍接受的成纤维细胞标记来识别静止和活化成纤维细胞，成纤维细胞的研究一直受到阻碍。纤维细胞是异质细胞群，难以分离和特征化。提出的协议描述了三种不同的方法来丰富纤维细胞和肌纤维细胞从未受伤和受伤的小鼠心脏。使用标准可靠的协议来分离成纤维细胞，可以研究它们在平衡和纤维化调制中的作用。
Introduction
心脏成纤维细胞，中质源细胞，除了在平衡1期间维持心脏结构外，在维持心脏的电传导和机械力方面起着重要作用。损伤后，这些细胞被激活，膨胀，并产生细胞外基质（ECM）蛋白质2。许多临床前研究表明，成纤维细胞是维持受伤心脏3的结构完整性的关键细胞调节器，以及负责ECM蛋白无节制生产和沉积的主要作用细胞，导致硬性疤痕形成和心力衰竭4。纤维细胞是一组异质细胞，很难从亲纤维的不适应特性中剖析它们的恢复功能。最近，确定了心肌损伤后两种不同成纤维细胞亚型的功能异质性，表明可以隔离不同的纤维细胞亚型，并研究其在伤口愈合中的作用。
获得纯成纤维细胞种群对于界定其在修复和纤维化中的作用至关重要。然而，多重纤维细胞标记的存在，识别其他细胞类型，使得分离一个基本上纯成纤维细胞种群6具有挑战性。一些优雅的研究已经设计了巧妙的方法来分离心脏成纤维细胞从未受伤和受伤的心肌。最流行和成熟的方法来丰富成纤维细胞是通过选择性粘附后酶组织消化7。
此外，基于细胞表面抗原的成纤维细胞荧光激活细胞分拣（FACS）也已成功描述8。在这项研究中，在酶消化之后，从小鼠心脏对间皮细胞按系系阴性（林：Ter119+CD45+CD31+）和gp38阳性（gp38+）分类。−Gp38_ve细胞根据col1+1和其他间质标记的共同表达被确认为成纤维细胞。虽然大多数组织消化是在解剖出培养皿中的心室后完成的，但最近一项研究调查了左心室中直接注入针头酶以分离肌细胞和非肌细胞（包括成纤维细胞9）的研究。在这种情况下，纤维细胞通过选择性粘附分离。
该协议使用三种方法描述了成纤维细胞的分离和浓缩。第一种是一种已经确立的方法，即酶消化后有选择性地粘附成纤维细胞。第二种方法主要用于主要分离损伤引起的阿尔法平滑肌肉表达肌纤维细胞。第三种方法涉及血液细胞和内皮细胞酶消化的心脏细胞悬浮液的连续磁耗竭。耗竭后，纤维细胞/肌纤维细胞根据抗原MEFSK4的存在而使用磁珠进行分离。最近，MEFSK4被描述为一种抗原，存在于静止和活化成纤维细胞上，使其成为纤维细胞识别和隔离的合适标记。当然，这里描述的所有方法都有独特的局限性。因此，强烈建议通过流量分析、免疫染色和半定量实时PCR来检查分离细胞群的纯度。然而，这些方法可以扩大，并添加额外的标记，以排除其他污染人群之前，利用成纤维细胞和肌纤维细胞种群进行关键的实验。
Protocol
这项研究严格坚持《国家卫生研究院实验室动物护理和使用指南》中的建议。范德比尔特大学机构动物护理和使用委员会批准了该协议（协议号：M1600076-01）。 1. 心脏解剖 解决方案准备 KHB 缓冲器 使用搅拌棒，在 900 mL DDI 水中缓慢溶解 9.4 克克雷布斯-亨塞莱特缓冲液 （KHB） 粉末。注：如果搅拌速度过快或搅拌时间过长，缓冲器会沉淀。KH…
Representative Results
使用 βSMA-GFP 报告小鼠显示肌纤维细胞分离的流量浇注方案未受伤的心脏在βSMA-GFP报告鼠模型中显示没有可检测的GFP+细胞;因此，它们被用来为补偿后GFP通道的背景信号建立一个门（图2）。*SMA=细胞根据在MI后10天从受伤左心室的GFP表达情况排序。一小部分内皮细胞（GFP+/CD31+） 细胞;SD = 3.8% = 0.0164;n = 5） 和造血性 （GFP+/CD45+ 细胞）;SD = 3.18% = 0.01…
Discussion
纤维细胞是一个异质细胞组，由不同的标记组识别。用于识别成纤维细胞的蛋白质标记物是二叶素域受体2（DDR2）、纤维素、维他丁、胶原蛋白I和III，以及Thy1 15、16、17、18、19、20。15,16,17,18,19,20维门汀已用于识别未受伤的静止心脏成纤维细胞，成?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢伊沃·卡拉伊齐奇博士对βSMA-GFP小鼠的感谢。本出版物中报告的研究得到了国家卫生研究院国家普通医学研究所（NIH）的支持，编号为R01GM118300（S.S.），国家国家卫生研究院生物医学成像和生物工程研究所根据编号 R21EB019509 （P.P.Y.） 和美国心脏协会的科学家发展补助金，奖励编号 17SDG33630187 （S.S.）。流动细胞测量分析在VUMC流细胞测量共享资源处进行，该资源由范德比尔特英格拉姆癌症中心（P30 CA68485）和范德比尔特消化疾病研究中心（DK0058404）支持。
Materials
| Reagents | |||
| Acetone | |||
| Anti-fungal (Amphotericin B-solubilized; Fungizone) | Sigma Aldrich | A9528 | |
| Bovine Serium Albumin (BSA) | Sigma | 9048-46-8 | |
| Calcium chloride | |||
| Citrate Buffer | |||
| Collagenase blend (Liberase Blendzyme 3 TH) | Roche Applied Science | ||
| DAPI | |||
| DDI water | |||
| DI water | |||
| DMEM-F12 with L-Glutamine and HEPES | Life technologies | 11330057 | |
| Dnase I(20U/mL) | BioRad | 7326828 | |
| Dulbecco's Phosphate-Buffered Saline (dPBS) without Ca2+ and Mg2+ | Gibco | 13190-144 | |
| 70% Ethanol | |||
| FC Blocker (Purified anti-mouse CD16/CD32) | Tonbo Biosciences | 70-0161 | |
| Fetal Bovine Serum (FBS) | Life technologies | 16000044 | |
| 10% goat serum | |||
| Hank's Balanced Salt Solution (HBSS) with Ca2+ and Mg2+ | Corning | 21-023-CV | |
| 1M HEPES | Corning | 25-060-Ci | |
| Krebs-Henseleit Buffer powder | Sigma | K3753 | |
| Mycoplasma prophylactic (Plasmocin) | Invivogen | ant-mpp | |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 15140122 | |
| 1x Phosphate-Buffered Saline (PBS) | |||
| 10x Red Blood Cell Lysis Buffer | Miltenyi | 130-094-183 | |
| Slow-fade Mounting Media | |||
| Sodium azide | |||
| Sodium bicarbonate | |||
| TGFβ | |||
| Trypan Blue Stain (0.4%) | Gibco | 15250-061 | |
| Type 1 Rat Collagen | |||
| Antibodies | |||
| 7AAD (stock: 1 mg/mL solution in DMSO) | Molecular Probes | A1310 | dilution = 1:1000; RRID = |
| CD45-APC | BD Bioscience | 559864 | dilution = 1:200; RRID = AB_398672 |
| CD31-PE | BD Bioscience | 553373 | dilution = 1:200; RRID = AB_394819 |
| CD31 | BD Biosciences | 553370 | dilution = 1:250; RRID = AB_394816 |
| CD45 | BD Biosciences | 553076 | dilution = 1:250; RRID = AB_394606 |
| COL 1α1 | MD Bioproducts | 203002 | dilution = 1:1000; RRID = |
| Ghost dye violet 510 (Formulation: 1 uL/test in DMSO) | Tonbo Biosciences | 13-0870 | dilution = 1:1000; RRID = |
| Goat anti-mouse Alexa Fluor 488 | Molecular Probes | A11029 | dilution = 1:200; RRID = AB_138404 |
| Goat anti-rabbit-Cy3 | Southern Biotech | 4050-02 | dilution = 1:200; RRID = AB_2795952 |
| Goat anti-rabbit-FITC | Jackson Immunoresearch Laboratories | 711-165-152 | dilution = 1:200; RRID = AB_2307443 |
| Goat anti-rat Alexa Fluor 488 | Molecular Probes | A11006 | dilution = 1:200; RRID = AB_2534074 |
| Goat anti-rat Alexa Fluor 647 | Thermo-Fisher | A21247 | dilution = 1:200; RRID = AB_141778 |
| Periostin | Santa Cruz | SC67233 | dilution = 1:100; RRID = AB_2166650 |
| Vimentin | Sigma Aldrich | V2258 | dilution = 1:200; RRID = AB_261856 |
| α-smooth muscle actin (αSMA) | Sigma Aldrich | A2547 | dilution = 1:1000; RRID = AB_476701 |
| Fibroblast specific protein 1 (FSP1) | Millipore 07-2274 | 07-2274 | dilution = 1:100; RRID = AB_10807552 |
| CD45 Magnetic Beads | Miltenyi Biotec | 130-052-301 | |
| CD31 Magnetic Beads | Miltenyi Biotec | 130-087-418 | |
| Anti-feeder cells-APC (MEFSK4) | Miltenyi Biotec | 130-102-900 | dilution = 1:100; RRID = AB_2660619 |
| anti-APC Beads | Miltenyi Biotec | 130-090-855 | |
| Rat IgG-APC | Miltenyi Biotec | 130-103-034 | dilution = 1:100; RRID = AB_2661598 |
| Donkey anti-rat Alexa Fluor405 | Abcam | ab175670 | dilution = 1:100 |
| anti-AN2/NG2 | Miltenyi Biotec | 130-097-455 | dilution = 1:11; RRID = AB_2651235 |
| Other Materials | |||
| 0.22 µm Filter | Thermo Scientific | 723-2520 | |
| 10 cm2 Cell Culture Dish | Corning | 430167 | |
| 10 mL Pipet | Fisherbrand | 13-678-11E | |
| 40 µm Cell Strainer | Fisherbrand | 22363547 | |
| 5 mL Pipet | Fisherbrand | 13-678-11D | |
| 50 mL Conical Tube | Falcon | 352070 | |
| 6-well Plate | Corning | 3506 | |
| Flow Cytometry Tubes | Falcon | 352058 | |
| Forceps | |||
| Rocker | |||
| Single Edge Blade | PAL | 62-0177 | |
| Surgical Scissors | |||
| GFP-αSMA Reporter Mice | |||
| MACS Separator Magnetic Field | |||
| MACS Separation Column | |||
| Coverslips | |||
| Qaigen Rneasy Mini Kit | Qaigen | 74104 | |
| Ambion RNAqueous Micro Total Isolation Kit | Ambion | AM1931 | |
| BioRad iScript cDNA Syntehsis Kit | BioRad | 1708891 | |
| 48-well Plate | |||
| 30G Needle | |||
| 3 Laser Flow Cytometry Machine (BD LSRFortessa) | BD Biosciences | ||
| 4 Laser Flow Cytometry Machine (BD FACSAria III) | BD Biosciences | ||
| Flow Data Acquiring Software (BD FACSDiva Software v8.0a) | BD Biosciences | ||
| Flow Data Analysis Software (FlowJo Software) | BD Biosciences |
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Citazione di questo articolo
Melzer, M., Beier, D., Young, P. P., Saraswati, S. Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts. J. Vis. Exp. (157), e60909, doi:10.3791/60909 (2020).