Keene D R, Maddox B K, Kuo H J, Sakai L Y, Glanville R W
Shriners Hospital for Crippled Children, Portland, Oregon 97201.
J Histochem Cytochem. 1991 Apr;39(4):441-9. doi: 10.1177/39.4.2005373.
High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.
使用含有6 M盐酸胍的缓冲液从人羊膜中提取高分子量聚集体。通过电子显微镜检查的旋转阴影制备物和负染色样品显示，每个聚集体似乎是由细丝连接的一串球状结构，呈现出串珠状外观。珠子的周期性是可变的。一种针对原纤维蛋白先前鉴定的胃蛋白酶片段的小鼠单克隆抗体与金标记的二抗一起用于免疫电子显微镜，以显示聚集体中含有原纤维蛋白。在皮肤、舌头、韧带、睫状小带、软骨和玻璃体液的非变性匀浆中也发现了类似的结构。当以与组织相同的方式制备用于电子显微镜的免疫金标记的串珠状结构时，不再能看到串珠状结构。相反，发现了金标记的微原纤维，其似乎与在结缔组织中观察到的含原纤维蛋白的基质微原纤维相同，并且通常与弹性蛋白相关。因此，与负染色或旋转阴影技术相比，包括固定、脱水和包埋在内的标准透射电子显微镜方案改变了微原纤维的超微结构外观。当皮肤在拉伸状态下仍处于张力下制备用于电子显微镜检查时，在组织切片中可见串珠状细丝，但在未拉伸的对照中不可见。此外，当拉伸的韧带在仍处于张力下用针对原纤维蛋白的抗体进行免疫标记时，与未拉伸的对照相比，沿着微原纤维的抗体周期性增加。我们提出，微原纤维包含由细丝连接的球状结构，这些细丝至少部分由高度有序、周期性分布的原纤维蛋白分子组成，其周期性会根据施加于包含它们的组织的张力而发生变化。
