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目的:探讨犬骨骼肌成肌细胞(SkMs)体外分离、纯化及培养方法的改良并研究其生物学特性。方法:采用机械分离结合Ⅱ型胶原酶、中性蛋白酶双酶一步消化法分离犬骨骼肌成肌细胞,经差速贴壁法纯化后,在骨骼肌细胞生长培养基(SKGM)中进行原代和传代培养,免疫细胞化学染色鉴定。结果:改良后的培养方法适于获取犬骨骼肌成肌细胞,SKGM培养基适于犬骨骼肌成肌细胞的体外培养。SkMs在细胞密集或低血清分化培养基作用下可融合成肌管。结蛋白(desmin)单克隆抗体(mAb)细胞化学染色鉴定SkMs呈阳性,纯度在90%以上。结论:通过改良后的双酶一步消化法获得的SkMs在合适的培养条件下能够增殖、分化并保持其生物学特性,为其在基因治疗和组织工程中的应用奠定了基础。
AIM: To investigate the isolation, purification and culture methods of skeletal muscle myoblasts (SkMs) in vitro and to study their biological characteristics. Methods: Skeletal muscle myoblasts were separated by mechanical separation combined with type Ⅱ collagenase and neutral protease digestion. After being purified by differential adherence method, primary culture was performed in skeletal muscle cell growth medium (SKGM) And subculture, immunocytochemical staining identification. Results: The improved culture method was suitable for obtaining canine myoblasts. SKGM medium was suitable for the culture of canine myoblasts in vitro. SkMs fuse to myotubes under the action of cell-dense or low-serum differentiation medium. Desmin monoclonal antibody (mAb) cytochemical staining SkMs positive, purity of more than 90%. CONCLUSIONS: SkMs obtained by the improved double-enzyme one-step digestion method can proliferate, differentiate and maintain their biological characteristics under appropriate culture conditions, which lays the foundation for their application in gene therapy and tissue engineering.