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

我们以前的冷冻断裂研究表明，Duchenne型肌营养不良症（DMD）肌质膜的特征之一是正交表显著减少。在未成熟肌纤维的质膜上也可见正交表的缺陷。因此，提出了批评，正交表的显着稀缺是由于DMD肌肉中再生纤维的存在。为了更精确地研究这个问题，我们打算通过快速冷冻、深蚀刻、旋转阴影（QDR）复制法同时观察肌质膜和肌丝，因为细胞质结构如肌原纤维排列暗示了肌纤维的成熟程度。取组织化学正常的6块人股四头肌和3例DMD患者的股四头肌，立即切成小块，用Eiko RF-23型快速冷冻装置，用金属接触法在氦气温度下快速冷冻。冷冻的肌肉样本 ...更多信息 转移到Eiko FD 5A冷冻断裂装置中，在-120 ° C和1-2 × 10 - 6托的真空下断裂<-7>，并在-90 ° C下深蚀刻30分钟。然后用电子束喷涂铂和碳，以30 °的阴影角旋转阴影。用漂白溶液消化肌肉组织，并将分离的复制品在蒸馏水中洗涤3次。电子显微镜的复制品显示清楚的肌纤维细胞骨架元素，如肌球蛋白和肌动蛋白丝的精细结构，但未能揭示正常肌肉质膜的正交阵列。本研究的结果表明，我们可以识别再生肌纤维的细胞质结构的外观，但正常肌肉质膜的超微结构中看到的复制品QDR方法是很大的不同，由传统的冷冻断裂方法，使用化学固定甘油正常人肌肉复制品。虽然我们复制了大量的正常人骨骼肌纤维，并通过电子显微镜进行了广泛的研究，我们不能找到任何正交阵列在其质膜。因此，本研究无法解决上述批评。然而，从这项研究中可以清楚地看出，QDR复制品方法最适合于观察肌纤维细胞骨架元素，如肌营养不良蛋白。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