Method for Decellularizing Skeletal Muscle Without Detergents or Proteolytic Enzymes
Method for Decellularizing Skeletal Muscle Without Detergents or Proteolytic Enzymes
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DOI：
10.1089/ten.tec.2010.0438
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发表时间：
2011-04-01
影响因子：
3
通讯作者：
Varghese, Shyni
中科院分区：
文献类型：
--
作者：
Gillies, Allison R.;Smith, Lucas R.;Varghese, Shyni
脱细胞骨骼肌是一种很有前途的模型，可用于研究肌病和肌肉萎缩性疾病中细胞-基质相互作用和肌肉细胞外基质（ECM）中发生的变化。本研究的目的是开发一种新的方法去细胞化骨骼肌，保持天然的生化成分和结构的ECM。该方法包括在latrunculin B、高离子强度盐溶液和DNA酶I中顺序孵育小鼠胫骨前肌，并且避免使用降解ECM的蛋白酶或去污剂。使用苏木精和伊红染色沿着DNA定量对脱细胞肌肉进行表征，表明DNA完全去除，而生化分析表明胶原蛋白没有损失，糖胺聚糖仅略有减少。脱细胞组织的蛋白质印迹分析表明去除了绝大多数的收缩蛋白肌动蛋白和肌球蛋白，并且使用扫描电子显微镜的形态学分析表明去除了脱细胞肌肉组织中的肌纤维。脱细胞肌束的被动力学测试显示了典型的非线性行为，类似于完整的肌肉。总之，这些结果表明，开发的方案成功地使骨骼肌脱细胞，而不改变其组成和机械功能。
Decellularized skeletal muscle is a promising model that can be used to study cell-matrix interactions and changes that occur in muscle extracellular matrix (ECM) in myopathies and muscle wasting diseases. The goal of this study is to develop a novel method to decellularize skeletal muscle that maintains the native biochemical composition and structure of the ECM. This method consists of sequential incubation of mouse tibialis anterior muscles in latrunculin B, high ionic strength salt solution, and DNase I and avoids use of proteases or detergents that degrade the ECM. Characterization of the decellularized muscles using hematoxylin and eosin staining along with DNA quantification suggested complete removal of DNA, whereas biochemical analyses indicated no loss of collagens and only a slight reduction in glycosaminoglycans. Western blot analysis of decellularized tissues showed removal of the vast majority of the contractile proteins actin and myosin, and morphological analysis using scanning electron microscopy suggested removal of myofibers from decellularized muscle tissues. Passive mechanical testing of decellularized muscle bundles revealed the typical nonlinear behavior, similar to that of intact muscle. Together, these results suggest that the protocol developed successfully decellularizes skeletal muscle without altering its composition and mechanical function.