从小鼠主动脉周围脂肪组织中分离、培养和诱导基质血管部分来源的前脂肪细胞
Summary
在这里，我们描述了从小鼠主动脉周围脂肪组织中分离、培养和诱导基质血管部分来源的前脂肪细胞，从而可以研究血管周围脂肪组织功能及其与血管细胞的关系。
Abstract
血管周围脂肪组织 （PVAT） 是一种围绕血管的脂肪组织库，表现出白色、米色和棕色脂肪细胞的表型。最近的发现揭示了PVAT在调节血管稳态和参与心血管疾病发病机制中的核心作用。全面了解PVAT特性和调控对于未来疗法的开发非常重要。主动脉周围脂肪细胞的原代培养对于研究PVAT功能以及主动脉周围脂肪细胞与血管细胞之间的串扰很有价值。本文提出了一种经济可行的方案，用于从小鼠主动脉周围脂肪组织中分离、培养和诱导基质血管部分来源的前脂肪细胞，可用于 体外模拟脂肪生成或脂肪生成。该方案概述了用于培养年轻小鼠主动脉周围脂肪细胞的组织处理和细胞分化。该协议将为研究PVAT功能提供工作台侧的技术基石。
Introduction
血管周围脂肪组织 （PVAT） 是一种由成熟脂肪细胞和基质血管部分 （SVF） 混合物组成的血管周围结构，被认为通过其分泌组旁分泌与相邻血管壁相互作用1。作为血管稳态的关键调节因子，PVAT 功能障碍与心血管疾病的发病机制有关 2,3,4。脂肪细胞组织的 SVF 由几个预期的细胞群组成，包括内皮细胞、免疫细胞、间皮细胞、神经元细胞以及脂肪干细胞和祖细胞 （ASPC）5,6。众所周知，存在于脂肪组织SVF中的ASPC可以产生成熟的脂肪细胞5。SVF 被推断为 PVAT 中成熟脂肪细胞的关键来源。多项研究表明，PVAT-SVF 可以在特定的诱导条件下分化为成熟的脂肪细胞 6,7,8。
目前，从脂肪组织中分离SVF有两种分离系统，一种是酶消化，另一种是非酶9。酶法通常可提高有核祖细胞的产量10。迄今为止，SVF 在促进伤口愈合、泌尿生殖系统和心血管疾病中的血管再生和新生血管形成方面的益处已得到广泛证明11，尤其是在皮肤病学和整形外科领域12,13。然而，PVAT衍生SVF的临床应用前景尚未得到很好的探索，这可能是由于缺乏标准化的SVF与PVAT分离方法。该协议的目的是建立一种标准化方法，用于从胸主动脉周围的小鼠PVAT中分离，培养和脂肪生成诱导SVF衍生的前脂肪细胞，从而能够进一步研究PVAT功能。该方案优化了组织加工和细胞分化技术，用于培养从年轻小鼠获得的主动脉周围脂肪细胞。
Protocol
动物用药方案经上海交通大学医学院附属上海胸科医院机构动物护理与使用委员会批准（批准文号：KS23010），符合相关伦理规范。4-8周龄的雄性和雌性C57BL / 6小鼠是该实验的首选。 1. 手术工具、缓冲液和培养基的制备 将手术工具（例如手术剪刀和标准镊子）在121°C高压灭菌30分钟。用75%酒精对显微外科器械进行消毒。 制备补充有 1% v/v 青霉素 – 链…
Representative Results
使用上述方案，我们仔细分离小鼠胸主动脉周围的PVAT（图1A-D）。使用无菌剪刀将PVAT洗涤并切碎成小块（图1E，F）后，将组织碎片在含有1型胶原酶（1mg / mL）和分散酶II（4mg / mL）的消化溶液中消化，并在37°C下在振荡器上孵育30-45分钟（图1G）。将消化的组织通过70μm细胞过滤器过滤到50mL离心…
Discussion
我们提出了一种实用可行的方法，用于从小鼠主动脉周围脂肪组织中分离和诱导SVF来源的前脂肪细胞。该协议的优点是简单且经济。足够数量的小鼠对于成功分离至关重要，因为组织不足会导致 SVF 密度低和生长状态不佳，最终影响脂肪生成效率。此外，小鼠年龄是一个需要考虑的重要因素，因为SVF的成脂潜力随着年龄的增长而降低。快速、仔细地分离PVAT，同时最大限度地减少脉管系统的污染至?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（82130012和81830010）和上海市胸科医院基础研究培育项目（批准号：2022YNJCQ03）的支持。
Materials
| 0.2 μm syringe filters | PALL | 4612 | |
| 12-well plate | Labselect | 11210 | |
| 15 mL centrifuge tube | Labserv | 310109003 | |
| 3,3',5-triiodo-L-thyronine (T3) | Sigma-Aldrich | T-2877 | 1 nM |
| 50 mL centrifuge tube | Labselect | CT-002-50A | |
| anti-adiponectin | Abcam | ab22554 | 1:1,000 working concentration |
| anti-COX IV | CST | 4850 | 1:1,000 working concentration |
| anti-FABP4 | CST | 2120 | 1:1,000 working concentration |
| anti-PGC1α | Abcam | ab191838 | 1:1,000 working concentration |
| anti-PPARγ | Invitrogen | MA5-14889 | 1:1,000 working concentration |
| anti-UCP1 | Abcam | ab10983 | 1:1,000 working concentration |
| anti-α-Actinin | CST | 6487 | 1:1,000 working concentration |
| BSA | Beyotime | ST023-200g | 1% |
| C57BL/6 mice aged 4-8 weeks of both sexes | Shanghai Model Organisms Center, Inc. | ||
| Cell Strainer 70 µm, nylon | Falcon | 352350 | |
| Collagen from calf skin | Sigma-Aldrich | C8919 | |
| Collagenase, Type 1 | Worthington | LS004196 | 1 mg/mL |
| Dexamethasone | Sigma-Aldrich | D1756 | 1 μM |
| Dispase II | Sigma-Aldrich | D4693-1G | 4 mg/mL |
| Fetal bovine serum | Gibco | 16000-044 | 10% |
| HEPES | Sigma-Aldrich | H4034-25G | 20 mM |
| High glucose DMEM | Hyclone | SH30022.01 | |
| IBMX | Sigma-Aldrich | I7018 | 0.5 mM |
| Incubator with orbital shaker | Shanghai longyue Instrument Eruipment Co.,Ltd. | LYZ-103B | |
| Insulin (cattle) | Sigma-Aldrich | 11070-73-8 | 1 μM |
| Isoflurane | RWD | R510-22-10 | |
| Krebs-Ringer's Solution | Pricella | PB180347 | protect from light |
| Microsurgical forceps | Beyotime | FS233 | |
| Microsurgical scissor | Beyotime | FS217 | |
| Oil Red O | Sangon Biotech (Shanghai) Co., Ltd | A600395-0050 | |
| PBS (Phosphate-buffered saline) | Sangon Biotech (Shanghai) Co., Ltd | B548117-0500 | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-035-146 | 1:5,000 working concentration |
| Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 111-035-144 | 1:5,000 working concentration |
| Rosiglitazone | Sigma-Aldrich | R2408 | 1 μM |
| Standard forceps | Beyotime | FS225 | |
| Surgical scissor | Beyotime | FS001 |
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Cite This Article
Liang, M., Huang, Y., Jiang, Y., Hu, Y., Cai, Z., He, B. Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue. J. Vis. Exp. (197), e65703, doi:10.3791/65703 (2023).