大鼠肌卫星细胞成肌分化的体外实验研究

THE STUDY OF RAT MYOGENIC SATELLITE CELLS DIFFERENTIATION TO MYOBLASTS IN VITRO

目的探讨肌卫星细胞体外分化和烟碱型乙酰胆碱受体表达。方法采用胰蛋白酶消化SD乳鼠的骨骼肌卫星细胞,差速贴壁法纯化,进行体外培养,观察卫星细胞的分化过程,利用免疫组织化学方法进行鉴定。用银环蛇毒素鉴定细胞表面的烟碱型乙酰胆碱受体,并用乙酰胆碱刺激细胞观察其收缩功能。结果体外培养的肌卫星细胞分化为成肌细胞,融合成肌管。α-sarcomeric actin、skeletal myosin和desmin抗体鉴定为阳性。ACh可刺激卫星细胞形成的肌管收缩。卫星细胞无明显的N-ACh受体表达;而卫星细胞形成的肌管经分化培养后N-ACh受体呈局部块状聚集。结论肌卫星细胞分化后形成的肌管具有收缩功能,并且在分化过程中逐渐合成烟碱型乙酰胆碱受体。

Objective To investigate the whole differentiating process of myogenic satellite cells in vitro, and the expression of N-ACh receptors. Methods Skeletal muscle satellite cells were isolated by trypsin digestion in vitro and purified by preferential attachment method. The cells were observed morphologically and identified by α-sarcomeric actin, desmin and skeletal myosin antibodies. Contraction of myotubes was observed after adding ACh in the culture medium, N-AChRs in myotube were also be identified by hungarotoxin. Results Cultured satellite cells in vitro differentiated into myohlasts and fused to form myotubes later. These cells were positive by α-sarcomeric actin, skeletal myosin and desmin antibodies＇ staining. Adding ACh directly in the medium stimulated the contraction of myotubes. No obvious expression of ACh receptors was observed on the surface of satellite cells. Cluster of receptors aggregated on myntubes from neonatal rats＇ satellite cells cultured in differentiation media. Conclusion The myntubes differentiated from myogenic satellite cells have the function of contraction. N-ACh receptors were synthesized gradually during the process of differentiation.