Mechanisms of skeletal muscle regeneration and factors affecting it.

研究了鸡背阔肌冷伤后再生纤维中膜下细胞骨架蛋白dystrophin和vinculin的表达和分布的时程变化。伤后2~7d，取ALD制备连续的横、纵向冰冻切片，分别用抗dystrophin、vinculin和心脏C蛋白(CCP)的抗体进行染色，然后与FITC标记的二抗和罗丹明标记的鬼臼蛋白孵育。随后在共聚焦显微镜下观察标本。用抗CCP抗体检测再生纤维。伤后3d开始出现CCP阳性再生纤维。此时，dystrophin在一些再生纤维的细胞膜上微弱而不均匀地分布。这些抗肌营养不良蛋白阳性的五个神经元中的一些对抗纽蛋白也有反应。损伤后7d，再生纤维呈不同程度的抗CCP染色反应，反映了再生的不同阶段。在纵切切片上，抗CCP染色较深的纤维含有稀疏分布的与鬼臼草素反应的肌动蛋白原纤维。在这些纤维的正面图中，dystrophin沿着细胞膜微弱而均匀地分布。中等水平的抗CCP阳性纤维除呈模糊弥漫分布外，还沿肌膜有一定程度的抗肌营养不良蛋白条纹分布。在抗CCP弱标记的纤维中，发达的肌原纤维充满细胞质，dystrophin显示出与完整纤维相同的明显的条纹和/或格子样图案。这些结果表明，在骨骼肌纤维再生过程中，dystrophin的表达早于vinculin，并且随着再生的进行，dystrophin在肌膜上的分布从均匀分布到条纹分布，再到网格状分布。

The time courses of the expression and distribution of submembranous cytoskeletal proteins, dystrophin and vinculin, were investigated in regenerating fibers of the anterior latissimus dorsi muscle (ALD) in the chicken after cold injury. At 2-7 days after injury, serial transverse and longitudinal frozen sections were prepared from the ALD,stained with antibodies against dystrophin, vinculin and cardiac C-protein (CCP) and then incubated with FITC-labeled secondary antibody and rhodamine-labeled phalloidin. The specimens were subsequently observed under a confocal microscope. The antibody against CCP was used to detect regenerating fibers. CCP-positive regenerating fibers first appeared at 3 days after injury. At this time, dystrophin was faintly and nonuniformly distributed along the cell membrane of some regenerating fibers. Some of these dystrophin-positive fivers were also reactive to anti-vinculin. At 7 days after injury, regenerating fibers exhibited various degrees of anti-CCP staining reactivity, reflecting a variety of regeneration stages. In longitudinal sections, fibers intensely stained with anti-CCP were found to contain sparsely distributed thin actin fibrils (miofibrils) reactive with phalloidin. In en face views of such fibers, dystrophin was faintly and uniformly distributed along the cell membrane. Fibers positive with anti-CCP at moderate levels exhibited a striated pattern of dystrophin to some extent along the sarcolemma in addition to the faint and diffuse distribution. In fibers weakly labeled with anti-CCP,well-developed myofibrils filled the cytoplasm, and dystrophin was found to exhibit distinct striations and/or a lattice-like pattern as those in intact fibers. These results suggest that dystrophin is expressed earlier than vinculin during regeneration of skeletal muscle fibers and that the distribution of dystrophin along the sarcolemma changes from the uniform to the striated, and to the lattice-like pattern as the regeneration advances.