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

人类营养不良肌肉的肌肉质膜和肌丝的冷冻蚀刻电子显微镜研究。

基本信息

批准号：
62570370
负责人：
WAKAYAMA Yoshihiro
金额：
$ 1.22万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

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

我们以前的冻结研究表明，杜钦肌营养不良症（DMD）的肌肉质膜的特征之一是正交阵列的显着下降。在未成熟的肌纤维的质膜中还可以看到正交阵列的缺陷。因此，批评主义提出了正交阵列的明显稀缺性是由于DMD肌肉中存在再生纤维所致。为了更准确地研究这个问题，我们打算仅通过快速冻结，深蚀刻，旋转阴影（QDR）复制方法观察肌肉质膜和肌膜，因为细胞质结构（例如肌原纤维排列）刺激了肌纤维的成熟程度。组织化学上正常的6个人股四头肌股肌肉和来自3例DMD患者的股股骨肌肉肌肉立即被切成小块，并通过金属接触方法在氦气温度下迅速冷冻，使用EIKO RF 23 Rapid Freeze设备。冷冻的肌肉样品……更多被转移到eiko FD 5A冻结裂缝设备中，并在-120 ﾟC下裂缝，真空为1-2 x 10^<-7> torr，并在-90°C下深蚀刻30分钟。然后将样品以30 ﾟ的阴影角度呈旋转，电子束枪声铂和碳。通过漂白溶液消化肌肉组织，并在蒸馏水中将分离的复制品洗涤3次。复制品的电子显微镜清楚地表明了肌纤维细胞骨架元素的精细结构，例如肌球蛋白和肌动蛋白丝，但未能揭示正常肌肉质膜中正交阵列。这项研究的结果表明，我们可以通过细胞质结构的出现来鉴定重生的肌纤维，但是正常肌肉质膜膜膜的超微结构在QDR方法中所作的复制品中，与使用化学固定的透明透明透明的正常人类肌肉制成的复制品相当不同于复制品的复制品。尽管我们制作了许多正常人骨骼肌肌纤维的复制品，并通过电子显微镜进行了广泛的检查，但我们在其质膜上找不到任何正交阵列。因此，我们无法在这项研究中解决上述批评主义。但是，从这项研究中可以清楚地看出，QDR副本方法最适合观察肌纤维细胞骨架元素，例如肌营养不良蛋白。较少的