Hilenski L L, Terracio L, Sawyer R, Borg T K
Department of Pathology, University of South Carolina, Columbia 29208.
Scanning Microsc. 1989 Jun;3(2):535-48.
The distribution and three-dimensional relationship of myofibrillar and cytoskeletal components during myofibrillogenesis were examined in preparations of neonatal rat cardiac myocytes processed in parallel for scanning electron microscopy (SEM), intermediate voltage transmission electron microscopy (IVEM) and immunofluorescence (IF). Of the various methods used for processing, optimal results were achieved by pre-extraction with Triton X-100 in an actin-stabilizing buffer. This procedure effectively removed the surface membrane, as viewed by SEM images, while preserving myofibrillar and cytoskeletal structure, as evidenced by IF for actin, alpha-actinin and vinculin. Cytoskeletons in SEM images consisted of a cortex of anastomosing filaments through which ran parallel filament bundles oriented in the long axis of the cell and attached along their length to the substrate by numerous fine filaments. In IVEM images, myofibrils were laterally connected at the level of the Z bands. Myocytes grown on different extracellular matrices showed different patterns and distributions of both striated myofibrils and focal adhesions, as determined by IF for alpha-actinin and vinculin, respectively. Cells on collagen I and III contained striated myofibrils which extended to the cell perimeters where focal adhesions were predominately located. Cells on laminin and fibronectin matrices exhibited myofibrils and focal adhesions more centrally located. In addition, cells on laminin contained circumferential arcs of filaments near the cell periphery.
在同时采用扫描电子显微镜（SEM）、中压透射电子显微镜（IVEM）和免疫荧光（IF）处理的新生大鼠心肌细胞制剂中，研究了肌原纤维生成过程中肌原纤维和细胞骨架成分的分布及三维关系。在所用的各种处理方法中，通过在肌动蛋白稳定缓冲液中用Triton X - 100进行预提取可获得最佳结果。如SEM图像所示，该程序有效去除了表面膜，而肌动蛋白、α - 辅肌动蛋白和纽蛋白的IF结果表明，肌原纤维和细胞骨架结构得以保留。SEM图像中的细胞骨架由相互吻合的细丝组成的皮质构成，平行的细丝束穿过该皮质，细丝束沿细胞长轴排列，并通过许多细丝沿其长度附着于底物。在IVEM图像中，肌原纤维在Z带水平横向连接。分别通过α - 辅肌动蛋白和纽蛋白的IF测定，在不同细胞外基质上生长的心肌细胞显示出横纹肌原纤维和粘着斑的不同模式和分布。在I型和III型胶原上的细胞含有延伸至细胞周边的横纹肌原纤维，粘着斑主要位于此处。在层粘连蛋白和纤连蛋白基质上的细胞，其肌原纤维和粘着斑更多地位于中央。此外，在层粘连蛋白上的细胞在细胞周边附近含有细丝的圆周弧。
