骨骼肌成纤维细胞外泌体的分离和表征
Summary
该方案说明了1）从成年小鼠腓肠肌中分离和培养原代成纤维细胞，以及2）使用差异超速离心法结合蔗糖密度梯度，然后进行蛋白质印迹分析的外泌体纯化和表征。
Abstract
外泌体是几乎所有细胞释放并在所有生物体液中分泌的小细胞外囊泡。已经开发了许多用于分离这些囊泡的方法，包括超速离心、超滤和体积排阻色谱法。然而，并非所有方法都适合大规模外泌体纯化和表征。这里概述了一种方案，用于建立从成年小鼠骨骼肌中分离的原代成纤维细胞的培养物，然后从这些细胞的培养基中纯化和表征外泌体。该方法基于使用连续离心步骤，然后是蔗糖密度梯度。然后使用一组经典标记物（即 Alix、CD9 和 CD81）通过蛋白质印迹分析来验证外泌体制剂的纯度。该方案描述了如何分离和浓缩生物活性外泌体，用于电子显微镜、质谱和功能研究的摄取实验。它可以很容易地放大或缩小，并适用于从不同细胞类型、组织和生物体液中分离外泌体。
Introduction
外泌体是异质细胞外囊泡，大小范围为 30-150 nm。鉴于它们在组织和器官中无处不在的分布，它们是生理和病理过程中的关键参与者 1,2。外泌体携带蛋白质、脂质、DNA 类型和 RNA 类型的复杂货物，这些货物根据它们来源的细胞类型而变化 1,2,3。外泌体富含具有不同功能的蛋白质（即四跨膜蛋白，包括 CD9 和 CD63）负责融合事件。例如，热休克蛋白 HSP70 和 HSP90 参与抗原结合和呈递。此外，Alix、Tsg101 和 flotillin 参与外泌体生物发生和释放，并被广泛用作这些纳米囊泡的标志物 2,3,4。
外泌体还含有多种 RNA（即 microRNA、长链非编码 RNA、核糖体 RNA），它们可以转移到受体细胞中，从而影响下游信号转导3。外泌体被单个单元膜包围，其生物活性不仅取决于蛋白质和核酸的货物，还取决于限制膜1的脂质成分。外泌体膜富含磷脂酰丝氨酸、磷脂酸、胆固醇、鞘磷脂、花生四烯酸和其他脂肪酸，所有这些都会影响外泌体稳定性和膜拓扑结构 2,3。由于货物和脂质排列，外泌体在接收细胞中启动信号通路并参与维持正常组织生理学 1,2,4,5。在某些病理条件下（即神经退行性变、纤维化和癌症），它们已被证明会触发和传播病理刺激 4,6,7,8,9,10,11。
由于外泌体能够将信号传播到邻近或遥远的部位，因此已成为诊断或预后疾病状况的有价值的生物标志物。此外，外泌体已被实验用作治疗化合物的载体 2,12。这些纳米囊泡在临床上的潜在应用使得分离方法变得越来越重要，以实现最大的产量、纯度和可重复性。已经开发和实施了用于分离外泌体的不同技术。通常，可以通过差速离心、体积排阻色谱和免疫捕获（使用市售试剂盒）从条件的细胞培养基或体液中分离外泌体。每种方法都有独特的优点和缺点，前面已经讨论过1、2、13、14。
概述的方案侧重于1）从成年小鼠腓肠肌中分离和培养原代成纤维细胞，以及2）这些细胞释放到培养基中的外泌体的纯化和表征。目前缺乏从原代成纤维细胞中分离外泌体以进行功能研究的成熟方案。原代成纤维细胞不分泌大量外泌体，使分离和纯化过程具有挑战性。该方案描述了从大培养体积中纯化大量纯外泌体，同时保持其形态完整性和功能活性。从条件培养基中获得的纯化外泌体已成功用于体外摄取实验，以诱导受体细胞中的特异性信号通路。它们还被用于对来自多个生物样品的外泌体货物进行比较蛋白质组学分析4。
Protocol
Representative Results
Discussion
如本协议所述，从培养基中成功分离外泌体的关键步骤是从成人骨骼肌中正确建立和维持原代小鼠成纤维细胞培养物。这些培养物需要保持在低氧水平，以确保生理条件（骨骼肌中的 O2 水平为 ~2.5%）15。原代成纤维细胞在培养中传承过多时会改变特征。因此，低传代数对于理想的外泌体产量是必不可少的。纯化的外泌体应立即用于实验目的，或在-80°C下以等分试样冷冻?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
亚历山德拉·达佐（Alessandra d’Azzo）是儿童珠宝商协会（JFC）遗传学和基因治疗的主席。这项工作得到了美国国立卫生研究院（NIH）拨款R01GM104981、RO1DK095169和CA021764、孟菲斯阿西西基金会和美国黎巴嫩叙利亚联合慈善机构的部分支持。
Materials
| 10 cm dishes | Midwest Scientific, TPP | TP93100 | |
| 15 cm dishes | Midwest Scientific | TP93150 | |
| BCA protein assay kit | Thermo Fisher Scientific, Pierce | 23225 | |
| Bovine serum albumin Fraction V | Roche | 10735094001 | |
| CaCl2 | Sigma | C1016-100G | |
| Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11 | Eppendorf | 022620659/5427754008 | |
| Chemidoc MP imaging system | BioRad | 12003154 | |
| Collagenase P | Sigma, Roche | 11 213 857 001 | 100 mg |
| cOmplete protease inhibitor cocktail | Millipore/Sigma, Roche | 11697498001 | |
| Criterion Blotter with plate electrodes | BioRad | 1704070 | |
| Criterion TGX stain-free protein gel | BioRad | 5678034 | 10% 18-well, midi-gel |
| Criterion vertical electrophoresis cell (midi) | BioRad | NC0165100 | |
| Dispase II | Sigma, Roche | 04 942 078 001 | neutral protease, grade II |
| Dithiothreitol | Sigma/Millipore, Roche | 10708984001 | |
| Dulbecco’s Modification Eagles Medium | Corning | 15-013-CV | |
| Dulbecco’s Phosphate Buffered Saline | Corning | 21-031-CV | |
| Ethanol 200 proof | Pharmco by Greenfield Global | 111000200 | |
| Falcon 50 mL conical centrifuge tubes | Corning | 352070 | |
| Fetal Bovine Serum | Gibco | 10437-028 | |
| Fluostar Omega multi-mode microplate reader | BMG Labtech | ||
| GlutaMAX supplement | Thermo Fisher Scientific, Gibco | 35050-061 | |
| Hydrochloric acid | Fisher Scientific | A144S-500 | |
| Immobilon-P Transfer membranes | Millipore | IPVH00010 | |
| Laemmli sample buffer (4x) | BioRad | 1610747 | |
| Magnesium acetate solution | Sigma | 63052-100ml | |
| Non-fat dry milk | LabScientific | M-0842 | |
| O2/CO2 incubator | Sanyo | MC0-18M | |
| Penicillin-Streptomycin | Thermo Fisher Scientific, Gibco | 15140-122 | 10,000 U/ml |
| Premium microcentrifuge tubes | Fisher Scientific, Midwest Scientific | AVSC1510 | 1.7 mL |
| Protected disposable scalpels | Fisher Scientific, Aspen Surgical Bard-Parker | 372610 | |
| Running buffer | BioRad | 1610732 | |
| Sodium Chloride | Fisher Scientific, Fisher Chemical | S271-3 | |
| Stericup Quick release-GP sterile vacuum filtration system | Millipore | S2GPU05RE | 500 mL |
| Sterile cell strainer (70 mm) | Fisher Scientific, Fisher brand | 22-363-548 | |
| Sucrose | Fisher Scientific, Fisher Chemical | S5-500 | |
| SuperSignal west Femto | Thermo Fisher Scientific | 34096 | |
| Thin wall Polypropylene tubes | Beckman Coulter | 326823 | |
| Transfer buffer | BioRad | 16110734 | |
| Trichloroacetic Acid | Sigma | 91228-100G | |
| Tris base | BioRad | 1610719 | |
| Triton-X100 solution | Sigma | 93443-100mL | |
| TrypLE Express Enzyme | Thermo Fisher Scientific, Gibco | 12604-013 | No phenol red |
| Tween-20 | BioRad | #1610781 | |
| Ultra-centrifuge Optima XPM | Beckman Coulter | A99842 | |
| Ultra-clear tube (14×89 mm) | Beckman Coulter | 344059 | |
| Ultra-clear tubes (25×89 mm) | Beckman Coulter | 344058 | |
| Water bath Isotemp 220 | Fisher Scientific | FS220 |
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