小鼠骨骼肌肉外泄原发性肌基器的分离和分化
Summary
肌细胞是增殖前体细胞，分化形成多核肌管，并最终骨骼肌肌纤维。在这里，我们提出了一个协议，从年轻的成年小鼠骨骼肌肉有效隔离和培养原发性肌体。该方法支持对培养中的肌肉细胞进行分子、遗传和代谢研究。
Abstract
原发性肌体是骨骼肌的无差别增殖前体。它们可以作为肌肉前体培养和研究，或诱导分化到肌肉发育的后期阶段。此处提供的协议描述了一种强大的方法，用于从年轻的成年小鼠骨骼肌外植体中分离和培养高度增殖的肌细胞群。这些细胞可用于研究不同小鼠模型骨骼肌的代谢特性，以及其他下游应用，如外源DNA转染或病毒表达载体转导。这些细胞的分化水平和代谢特征取决于暴露的长度，以及用于诱导月细胞分化的介质的组成。这些方法为研究小鼠肌肉细胞外体代谢提供了强有力的系统。重要的是，与体内模型不同，本文描述的方法提供了一个细胞群，可以扩展和研究具有高重复性。
Introduction
虽然经常被引证为整体代谢健康的指示，但多项研究表明，老年人的身体质量指数（BMI）与较高的死亡率风险并不一致。迄今为止，唯一与降低这一种群死亡率相一致的因素是增加肌肉质量1。肌肉组织是体内胰岛素敏感细胞的最大来源之一，因此对维持整体代谢平衡至关重要。通过运动激活骨骼肌组织与增加局部胰岛素敏感性和整体代谢健康3相关。虽然体内模型对于研究肌肉生理学和肌肉功能对综合代谢的影响至关重要，但肌管的主要培养物提供了一个可处理的系统，可降低动物研究的复杂性。
从产后肌肉衍生的肌末代酶可用于以高度可重复的方式研究多种治疗和生长条件的影响。这早已得到承认，几种方法的月经隔离和文化已经描述了4，5，6，7，8，9。其中一些方法使用新生儿肌肉，产生相对较少的肌体5，8，需要几种动物进行更大规模的研究。此外，大多数广泛使用的培养霉细胞的方法使用”预镀”来丰富于于其他细胞类型粘附性较差的细胞。我们发现这里描述的替代富集方法对于丰富高度增殖的美洲人群体更有效和可重复。总之，该协议允许从年轻的成人肌肉外植分离高增殖性肌体，通过生长到培养培养。月子可以反复收获，在几天内，迅速扩大，并诱导分化成近管。该协议可重复产生大量健康的肌细胞，这些细胞会强力分化成自发抽搐肌细胞。它使我们能够研究各种基因型小鼠的原发性月管的代谢和昼夜节律。最后，我们采用96孔板中耗氧率的测量方法，为氧化代谢研究制备肌管。
Protocol
该协议遵循斯克里普斯研究的动物护理指南。 1. 肌肉组织外泄的收集和处理 解剖前一天，对所有解剖设备（钳子、剃须刀和剪刀）进行消毒，并准备所有必需的介质：磷酸盐缓冲盐水（PBS）、甲基溴电镀介质（DMEM 12.5 mL、HAMS F12 12 12.12 mL、20 mL 热灭活胎牛血清 （FBS）， 5 mL 羊水介质补充剂） 和涂层溶液 （24 mL DMEM， 24 mL HAMS F12， 1.7 mL 胶原蛋白， 1 mL 母体凝胶）. …
Representative Results
按照所提供的协议第 1 节，应产生从植物外部产生的原细胞，这些细胞将在标准光显微镜下可见（图 2）。异质细胞群将生长在除外每个肌肉组织外部和周围。微球体将显示为小，圆，明亮的球体。根据协议第2节，将产生从组织外植的细胞细胞的早期收获，这将包含很少的细胞，并且将是异质的（图3）。该协议第3节描述了用PB…
Discussion
骨骼肌对于建立和维护代谢平衡11至关重要。肌肉生理学的研究因个体间变异性以及获取样本的困难而变得复杂，特别是在人类研究中。培养的原发性肌管已被证明可以重述肌肉生理学的许多特征，包括钙平衡12、受损肌肉组织的再生5、对运动13的代谢改变，糖尿病等疾病引起的新陈代谢的改变。小鼠的肌细…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢墨尔本大学的马修·瓦特博士和约翰霍普金斯大学的阿纳斯塔西娅·克拉利博士，他们根据莫克贝尔等人6号的工作，协助采纳这一协议。我们还感谢萨宾·乔丹博士协助在我们的实验室中制定和通过这一议定书。这项工作由美国国家卫生研究院R01s DK097164和DK112927资助。
Materials
| Coating Solution: | |||
| DMEM | Gibco | 10569010 | Always add gentamicin (1:1000 by volume) prior to use; 24 mL |
| HAMS F12 | Lonza | 12-615F | Always add gentamicin (1:1000 by volume) prior to use; 24 mL |
| Collagen | Life Technologies | A1064401 | 1.7 mL |
| Matrigel | Fisher | CB40234A | 1 mL |
| Plating Media: | |||
| DMEM | Gibco | 10569010 | Always add gentamicin (1:1000 by volume) prior to use; 12.5 mL |
| HAMS F12 | Lonza | 12-615F | Always add gentamicin (1:1000 by volume) prior to use; 12.5 mL |
| Heat Inactivated FBS | Life Technologies | 16000044 | 20 mL; can be purchased as regular FBS and heat-inactivated by placing in a 40 °C water bath for 20 minutes |
| Amniomax | Life Technologies | 12556023 | 5 mL |
| Myoblast Media: | |||
| DMEM | Gibco | 10569010 | Always add gentamicin (1:1000 by volume) prior to use; 17.5 mL |
| HAMS F12 | Lonza | 12-615F | Always add gentamicin (1:1000 by volume) prior to use; 17.5 mL |
| Heat Inactivated FBS | Life Technologies | 16000044 | 10 mL; can be purchased as regular FBS and heat-inactivated by placing in a 40 °C water bath for 20 minutes |
| Amniomax | Life Technologies | 12556023 | 5 mL |
| Differentiation Media: | |||
| DMEM | Gibco | 10569010 | Always add gentamicin (1:1000 by volume) prior to use; 24 mL |
| HAMS F12 | Lonza | 12-615F | Always add gentamicin (1:1000 by volume) prior to use; 24 mL |
| Heat Inactivated Horse Serum | Sigma | H1138 | 1.5 mL |
| Insulin-Selenium-Transferrin | Life Technologies | 41400045 | 0.5 mL |
| Other Materials: | |||
| PBS | Gibco | 14040133 | |
| Gentamicin | Sigma | G1397 | |
| TrypLE | Gibco | 12604013 | |
| DMSO | Sigma | 472301 | Prepare as 10% DMSO in Myoblast Media for freezing cells |
| Forceps | Any | ||
| Razor Blades | Any | ||
| Scissors | Any | ||
| Whatman paper | VWR | 21427-648 | |
| 60 mm plate | VWR | 734-2318 | |
| 10 cm plate | VWR | 25382-428 (CS) | |
| T25 Flasks | ThermoFisher | 156367 | |
| T75 Flasks | ThermoFisher | 156499 | |
| Centrifuge Tubes (15mL) | BioPioneer | CNT-15 | |
| Oxygen Consumption Rates: | |||
| Seahorse XFe96 Analyzer | Agilent | Seahorse XFe96 Analyzer | Instrument used to measure oxygen consumption rates read out by acidification of the extracellular media |
| Seahorse XFe96 FluxPak | Agilent | 102416-100 | 96-well plates for use in XFe96 Analyzer |
| Seahorse XF Cell Mito Stress Test Kit | Agilent | 103015-100 | components may be purchased from other suppliers once assay is established; some recommendations are listed below |
| Seahorse XF Palmitate-BSA FAO substrate | Agilent | 102720-100 | components may be purchased from other suppliers once assay is established; some recommendations are listed below |
| Palmitic acid | Sigma | P5585-10G | for measurement of fatty acid oxidation |
| carnitine | Sigma | C0283-5G | for measurement of fatty acid oxidation |
| Etomoxir | Sigma | E1905 | for measurement of fatty acid oxidation |
| BSA | Sigma | A7030 | used as control or in conjugation with palmitic acid for use in measurement of fatty acid oxidation |
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Citazione di questo articolo
Vaughan, M., Lamia, K. A. Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants. J. Vis. Exp. (152), e60310, doi:10.3791/60310 (2019).