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Observation of dystrophin molecule in huma skeletal myofibers by elctron microscopy of quick freeze, deep etch, rotary shadow replicas.

通过快速冷冻、深蚀刻、旋转阴影复制品的电子显微镜观察人类骨骼肌纤维中的肌营养不良蛋白分子。

基本信息

批准号：
02807084
负责人：
WAKAYAMA Yoshihiro
金额：
$ 0.96万
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：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The aim of this st'udy is to observe the shape and distribution of dystrophin molecule in normal human skeletal myofiber by using quick-freeze, deep etch, rotary shadow replica method. Six histochemically normal human quadriceps femoris-muscles undergoing orthopedic operations and six Duchenne biopsied muscles were fixed in chilled 4% paraformaldehyde solution for 2 hours. After washing, the fixed muscles were cut into thin slices by cryostat and the sliced muscles were frozen quickly by liquid helium and then the samples were transferred into freeze fracture apparatus. Fracture was carried out in the vacuum 1, -2 x 10^<-7> Torr at -150ﾟC. The specimens were deep etched at -90ﾟC for 30 minutds, cooled up to -150ﾟC again and rotary shadowed by electron beam gunned platinum and carbon. Electron microscopy of the good quality replicas revealed the orderly arranged myofilaments and, at higher magnification, the myosin cross bridge and about 5nm periodicity of surface contour of actin filam … More ents. There were two modes of fracture and deep-etching faces in myofibers of both Duchenne and normal cbntkol muscles. In one mode, the line of splitting ran frord the basal lamina to the true outer surface bf the muscle plasma membrane, then-enteted the hydrophobic interior of the muscle plasma membrane and jumped into the interior of the myofibers. In this case, the piffotoplasmic fracture face of the muscle plasma membrane and adjacent subsarcolemmal filaments were seen. In the other mode of fracture, the line of splitting ran from the cytoplasm of the myofiberto the cytoplasmic surface of the plasma membrane, then entered the hydrophobic interor of the muscle plasma membrane and jumped into the exterior of the myofibers. In this case, the extracellular fracture face and adjacent cytoplasmic surface of the muscle plasma membrane were noted. At higher magnification, the attachment of individual rod-shaped cytoskeletons of various size to the cytoplasmic surface of the plasma membrane was observed and the elaborate network of cytoskeletal elements was observed in both Duchenne and normal control myofibers. Since th. e marked difference of shape and distribution pattern of individual muscle plasma membrane associated cytoskeletons-were detected between Duchenne and normal control myofibers, dystrophin will be detectecd in the normal muscle specimens labelled by antidystrophin antibodies. Less

本研究采用快速冷冻、深腐蚀、旋转阴影复制法观察正常人骨骼肌肌纤维中抗肌萎缩蛋白分子的形态和分布。将6块组织化学正常的人股四头肌和6块Duchenne活检肌肉固定在冷冻的4%多聚甲醛溶液中2小时。清洗后，用低温恒温器将固定的肌肉切成薄片，用液氦快速冷冻，然后将样品转移到冷冻断裂装置中。断裂在真空中在-150 ° C下进行，真空度为1，-2 X 10 μ <-7>Torr。试样在-90 ° C下深腐蚀30分钟，再次冷却至-150 ° C，并用电子束喷涂铂和碳旋转阴影。电镜下可见肌丝排列整齐，高倍镜下可见肌球蛋白横桥，肌动蛋白丝表面轮廓周期性约为5 nm ...更多信息 ents。Duchenne肌和正常肌的肌纤维有两种断裂方式和深蚀面。在一种模式中，分裂线从基底膜到肌质膜的真实外表面，然后进入肌质膜的疏水内部并跳跃到肌纤维的内部。本例可见肌细胞膜及邻近肌膜下纤维的裂面。在另一种断裂方式中，断裂线从肌纤维的细胞质延伸到质膜的细胞质表面，然后进入肌质膜的疏水性内部，并跳跃到肌纤维的外部。在这种情况下，细胞外骨折面和相邻的肌质膜的细胞质表面被注意到。在更高的放大倍数下，观察到不同大小的单个杆状细胞骨架附着在质膜的细胞质表面，并在杜氏肌纤维和正常对照肌纤维中观察到细胞骨架元素的精细网络。自从th。在Duchenne肌纤维和正常对照肌纤维中，单个肌质膜相关胞浆素的形态和分布模式存在显著差异，抗肌营养不良蛋白抗体标记的正常肌肉标本中可检测到抗肌营养不良蛋白。少