从小鼠骨骼肌分离I型和II型周细胞
Summary
这项工作描述了基于FACS的协议，允许从骨骼肌容易和同时地分离I型和II型周细胞。
Abstract
周细胞是血管内多能细胞，其表现出不同器官甚至在相同组织内的异质性。在骨骼肌中，至少有两个周细胞亚群（称为I型和II型），其表达不同的分子标记并具有明显的分化能力。使用NG2-DsRed和Nestin-GFP双转基因小鼠，成功分离了I型（NG2-DsRed + Nestin-GFP）和II型（NG2-DsRed + Nestin-GFP + ）周细胞。然而，这些双转基因小鼠的可用性阻止了该纯化方法的广泛使用。这项工作描述了一种替代方案，允许从骨骼肌容易和同时隔离I型和II型周细胞。该协议采用荧光激活细胞分选（FACS）技术，并将靶蛋白GFP信号靶向PDGFRβ而不是NG2。隔离后，键入I和type II周细胞显示不同的形态。此外，用这种新方法分离的I型和II型周细胞，如分离自双转基因小鼠的那些，分别是脂肪形成和肌原性的。这些结果表明，该方案可用于从骨骼肌和可能来自其他组织中分离周细胞亚群。
Introduction
肌营养不良是一种肌肉退行性疾病，目前尚无有效治疗。促进组织再生的疗法的发展一直是非常有意义的。损伤后组织再生和修复取决于常驻干细胞/祖细胞1 。卫星细胞承诺造血前体细胞有助于肌肉再生2,3,4,5,6,7。然而，他们的临床使用受到其有限的迁移和注射后的低存活率以及在体外扩增后其分化能力降低8,9,10,11的阻碍。除了satellite细胞，骨骼肌也含有许多其他具有肌原性潜能12,13,14,15,16等细胞群，如血小板衍生生长因子受体-β（PDGFRβ） – 阳性间质细胞。有证据表明，肌肉衍生的PDGFRβ +细胞能够分化成肌原细胞，改善肌肉病理和功能14,17,18,19,20。 PDGFRβ主要标记周细胞21 ，它们是多能性的血管周围细胞22,23 。除PDGFRβ之外，许多其他标志物，包括神经元胶质细胞2（NG2）和CD146也被用于齿状细胞21 。然而，应该注意的是，这些标记物都不是percyte特异性21 。最近的研究揭示了肌肉周细胞的两种亚型，称为I型和II型，其表达不同的分子标记并实现不同的功能19,24,25。生物化学上，I型周细胞是NG2 + Nestin，而II型周细胞是NG2 + Nestin + 19,24。功能上，I型周细胞可能经历脂肪分化，有助于脂肪堆积和/或纤维化，而II型周细胞可以沿着肌原性途径分化，从而有助于肌肉再生19,24,25。这些结果表明：（1）I型周细胞可能be针对脂肪退化性疾病/纤维化的治疗，（2）II型周细胞对肌营养不良具有很大的治疗潜力。这些人群的进一步调查和表征需要能够以高纯度分离I型和II型周细胞的分离方案。
目前，周细胞亚群的分离取决于NG2-DsRed和Nestin-GFP双转基因小鼠19,24。 NG2-DsRed小鼠的可用性和大多数NG2抗体的质量限制了该方法的广泛使用。鉴于所有NG2 +周细胞也在骨骼肌19,20,24中表达PDGFRβ，我们假设NG2可被PDGFRβ替代以分离周细胞及其亚群。这项工作描述了基于FACS的协议使用PDGFRβ染色和Nestin-GFP信号。这种方法对于研究者来说要求较低，因为：（1）它不需要NG2-DsRed背景，（2）它使用市售的PDGFRβ抗体，其被良好表征。此外，它能够以高纯度同时分离I型和II型周细胞，从而进一步研究这些周细胞亚群体的生物学和治疗潜力。纯化后，这些细胞可以在培养物中生长，并且它们的形态可以被可视化。这项工作还表明，使用这种方法分离的I型和II型周细胞，如从双转基因小鼠纯化的那些，分别是脂肪形成和肌原性的。
Protocol
Representative Results
Discussion
周细胞是位于毛细血管21,26的外侧表面上的多能血管周围细胞22,23 。在骨骼肌中，周细胞能够沿着脂肪生成和/或造血途径19,20,24区分。最近的研究揭示了两个亚细胞周期细胞，具有不同的标记表达和不同的分化潜力19,24,25。 I型（NG2 + Nestin – ）周细胞是脂肪形成的，而II型（NG2 + Nestin + ）周细胞是肌原性的。这些亚群体外研究的分离需…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了强直性营养不良基金会（MDF-FF-2014-0013）和美国心脏协会（16SDG29320001）的科学家发展拨款的基金A-Fellow资助部分支持。
Materials
| Cell Sorter | Sony | SH800 | |
| Automatic Setup Beads | Sony | LE-B3001 | |
| DMEM | Gibco | 11995 | |
| Avertin | Sigma | T48402 | |
| Pericyte Growth Medium | ScienCell | 1201 | |
| MSC Basal Medium (Mouse) | Stemcell Technologies | 5501 | |
| Adipogenic Stimulatory Supplement (Mouse) | Stemcell Technologies | 5503 | |
| Fetal Bovine Serum | Gibco | 16000 | |
| Horse Serum | Sigma | H1270 | |
| Collagenase Type 2 | Worthington | LS004176 | |
| 0.25% Trypsin/EDTA | Gibco | 25200 | |
| Penicillin/Streptomycin | Gibco | 15140 | |
| PDL | Sigma | P6407 | |
| PDGFRβ-PE Antibody | eBioscience | 12-1402 | |
| Perilipin Antibody | Sigma | P1998 | |
| S-Myosin Antibody | DSHB | MF-20 | |
| Alexa 555-anti-rabbit antibody | ThermoFisher Scientific | A-31572 | |
| Alexa 555-anti-mouse antibody | ThermoFisher Scientific | A-31570 | |
| Mounting Medium with DAPI | Vector Laboratories | H-1200 | |
| DAPI | ThermoFisher Scientific | D1306 | |
| HEPES | Gibco | 15630 | |
| EDTA | Fisher | BP120 | |
| BSA | Sigma | A2058 | |
| NH4Cl | Fisher Scientific | A661 | |
| KHCO3 | Fisher Scientific | P184 | |
| PBS | Gibco | 14190 | |
| 18G Needles | BD | 305196 | |
| 10ml Serological Pipette | BD | 357551 |
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