新生SD大鼠成肌细胞体外培养的实验研究

Primary culture of myoblasts of neonatal rat

目的 :了解新生SD大鼠成肌细胞在体外培养条件下的生物学特性。方法 :新生 3d内SD大鼠幼崽 ,切取骨骼肌标本。应用Blau法经胰蛋白酶与胶原酶多步消化、分离成肌细胞。差速贴壁法纯化后 ,在 φ =2 0 %的胎牛血清生长培养基中培养 ,绘制生长曲线评价其增殖情况 ,φ =5 %的胎牛血清融合培养基培养 ,计算成肌细胞融合率评价其分化能力。通过光镜、免疫组织化学方法鉴定所得细胞。结果 :培养后成肌细胞免疫组织化学染色强阳性 ,能融合形成肌小管 ,证明为骨骼肌成肌细胞。在生长培养基中原代细胞倍增时间为 5d ,在融合培养基中肌小管融合率较高。结论 :多步酶消化法与差速贴壁法能获得足量、纯净的成肌细胞 ,所得细胞增殖力强 ,能表达骨骼肌特异收缩蛋白 ,融合培养基能促进其体外分化。

objective: To investigate the biological characteristics of neonatal SD rat skeletal myoblasts cultured in vitro . Methods: The skeletal muscle samples were obtained from neonatal SD rats younger than 3 days. With a modified method of Blau,the muscle samples were digested with trypsin and collagenase. The cell suspension was a mixture of myoblasts and fibroblasts, the latter were removed by repeated attachment to culture dishes. The characteristics of the cells were studied by morphological and immunohistochemical observation ,the proliferation was tested by cell counting. Results: The isolated myoblasts were spherical in cell suspension and spindle like after attached to culture dishes. The cells were myosin positive, when the cell density increased, the monocyte myoblasts could fused to form multinucleated myotube. The doubling time of the cells was 5 days. Conclusion: A large number of myoblasts can be obtained by enzyme digestion and repeated attachment method. The cultured cells can be proved as myoblasts by morphological and immunohistochemical detection.