Freeze etching electron microscopic study of muscle plasma membranes and myofilaments of human dystrophic muscles.

我们以前的冷冻骨折研究表明，Duchenne型肌营养不良症(Duchenne型肌营养不良症)的肌浆膜特征之一是正交表的显着减少。在未成熟肌纤维的质膜上也可以看到正交阵的缺陷。因此，批评人士提出，正交表的明显稀缺性是由于DMD肌肉中存在再生纤维。为了更准确地研究这个问题，我们打算用快速冷冻、深度蚀刻、旋转阴影(QDR)复制法同时观察肌质膜和肌丝，因为肌原纤维排列等细胞质结构暗示了肌纤维的成熟程度。组织化学正常的3例DMD患者的6块股四头肌和股四头肌，立即切成小块，用Eiko RF-23快速冰冻仪在氦气温度下快速冷冻。冰冻肌肉样本…将更多的材料转移到Eiko FD 5A冷冻断裂仪中，在-120ﾟC和1-2×10；-7托的真空度下断裂，并在-90ﾟC下深度蚀刻30分钟。然后用电子束喷射铂和碳，以30ﾟ的阴影角旋转阴影样品。肌肉组织用漂白液消化，分离的复制品用蒸馏水清洗3次。电子显微镜下可清楚地显示肌球蛋白、肌动蛋白细丝等肌纤维细胞骨架元素的精细结构，但未能显示正常肌肉质膜中的正交性排列。本研究结果表明，我们可以通过细胞质结构的外观来识别再生的肌纤维，但QDR法制作的正常肌质膜的超微结构与用化学固定的甘油处理的正常人肌肉用常规冷冻断裂法制作的复制件有很大的不同。虽然我们制作了大量正常人骨骼肌纤维的复制品，并用电子显微镜对其进行了广泛的观察，但在它们的质膜上没有发现任何正交表。因此，我们无法在本研究中解决上述批评。然而，从这项研究中可以清楚地看出，QDR复制法最适合于观察肌纤维细胞骨架元件，如肌营养不良蛋白。较少

Our previous freeze fracture studies showed that one of the characteristics of muscle plasma membrane of Duchenne muscular dystrophy (DMD) was the marked decrease of orthogonal arrays. The defect of orthogonal arrays is also seen in the plasma membrane of immature myofibers. Therefore the criticism raised that the marked scarcity of orthogonal arrays is due to the presence of regenerating fibers in DMD muscles. To investigate this problem more precisely, we intended to observe the muscle plasma membrane and myofilaments simultaneously by quick freeze, deep etch, rotary shadow (QDR) replica method, since the cytoplasmic structure such as myofibrillar arrangement hints the degree of maturation of myofibers. Histochemically normal 6 human quadriceps femoris muscles and the quadriceps femoris muscles from 3 patients with DMD were immediately cut into small pieces and rapidly frozen at helium temperature by metal contact method with Eiko RF 23 rapid freeze device. The frozen muscle samples … More were transferred to the Eiko FD 5A freeze fracture apparatus and fractured at -120ﾟC and a vacuum of 1-2 X 10^<-7> Torr, and deep etched at -90ﾟC for 30 minutes. Then the specimen were rotary shadowed at shadow angle of 30ﾟ by electron beam gunned platinum and carbon. The muscle tissues were digested by bleaching solution and the detached replicas were washed 3 times in distilled water. Electron microscopy of the replicas showed clearly the fine structure of myofiber cytoskeletal elements such as myosin and actin filaments, but failed to reveal the orthogonal arrays in normal muscle plasma membrane. The results of this study demonstrated that we could identify the regenerating myofibers by the appearance of cytoplasmic structure but the ultrastructure of normal muscle plasma membrane seen in the replicas made by QDR method was quite different from that of replicas made by conventional freeze fracture method using chemically fixed glycerinated normal human muscles. Although we made a lot of replicas of normal human skeletal myofibers and examined them extensively by electron microscope, we could not find any orthogonal arrays at their plasma membranes. Therefore we could not solve the above mentioned criticism in this study. However, it became clear from this study that the QDR replica method was most suitable for the observation of myofiber cytoskeletal elements such as dystrophin. Less