Immunoelectron microscopic and freeze etch studies of muscle plasma membrane and extracelular matrix in normal and diseased skeletal myofiber

用抗β-肌营养不良蛋白聚糖抗体和抗麦角蛋白单抗对人骨骼肌进行了免疫金电镜法研究。我们还打算通过使用抗α-肌营养不良蛋白聚糖抗体进行这项研究。根据已发表的α-肌营养不良聚糖基因序列，针对三个合成肽产生抗α-肌营养不良聚糖抗体(自然355：696-702,1992)。然而，我们的α-营养不良多糖抗体现在正在分析它们的特征。抗β-肌营养不良蛋白聚糖定位于骨骼肌纤维的肌膜下区域(J Electron Microsc 43：327-331,1994；Am J Pathol 146：189-196,1995)。相比之下，人骨骼肌纤维中Merosin的超微结构定位仅出现在肌质膜外和基底板成分的内侧。此外，冷冻蚀刻复制品的基板的电子显微镜显示，短的细胞骨架垂直于肌质膜表面，它与基板的这种小纤维结构的网状结构相连，该结构也含有较厚的IV型胶原纤维。Merosin信号定位于人体骨骼肌纤维复杂基板的内侧。该蛋白被证明是α-dystroglan受体(Sunada Y.et al.J Biol Chem 269：13729-13732,1994)，因此与金标记的merosin分子相关的细胞骨架元件被认为是与dystrophin virβ-dystroglan相连的α-dystroglan。

Immunogold electron microscopic investigation was carried out in human skeletal muscles by using anti beta-dystroglycan antibodies and anti merosin monoclonal antibody. We also intended to perform the study by using anti alpha-dystroglycan antibodies. Anti alpha-dystroglycan antibodies were generated against three synthetic peptides deduced on the published alpha-dystroglycan cDNA sequence (Nature 355 : 696-702,1992). However, our alpha-dystroglycan antibodies are now under analysis for their characterization. The anti beta-dystroglycan was ultrastructurally localized in the subsarcolemmal area of skeletal myofibers (J Electron Microsc 43 : 327-331,1994 ; Am J Pathol 146 : 189-196,1995). In contrast, the ultrastructural localization of merosin in human skeletal myofiber was present just outside of muscle plasma membrane and just inside area of basal lamina components. In addition, electron microscopy of basal lamina of freeze etched replica showed that the short cytoskeleton was standing perpendicular to the surface of muscle plasma membrane and it was connected to the network formation of this small fibrillar structures of basal lamina which also contained more thick collagen type IV fibrills. The signal of merosin was localized at the inside part of the complicated basal lamina of huma skeletal myofibers. The merosin was turned out to be the alpha-dystroglycan receptor (Sunada Y.et al.J Biol Chem 269 : 13729-13732,1994), so the cytoskeletal element associated with the gold labelled merosin molecules is thought to be the alpha-dystroglycan which is connected with dystrophin vir beta-dystroglycan.