Shapiro F, Cahill C, Malatantis G, Nayak R C
Department of Orthopaedic Surgery, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115.
Anat Rec. 1995 Jan;241(1):39-48. doi: 10.1002/ar.1092410107.
The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position.
The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2-3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures -30 degrees C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding.
Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intracellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them.
Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions.
采用免疫金标记技术和透射电子显微镜来证实中间丝波形蛋白在大鼠成骨细胞和骨细胞的细胞体及细胞突起中的表达和位置。对骨细胞进行的传统光学显微镜和透射电子显微镜研究表明，相邻细胞连接是由贯穿骨基质的小管系统内的成骨细胞和骨细胞突起介导的。细胞突起内充满了密集排列的细丝，其中许多细丝先前已被证明是肌动蛋白微丝。然而，在一些细胞突起中出现了直径为10纳米的细丝，并且中间丝波形蛋白已在许多间充质起源的细胞中被确定，这就增加了这些细丝中一些可能是波形蛋白的可能性。超微结构胶体金免疫化学技术能够原位证实波形蛋白细丝的表达并精确确定其位置。
研究在新生大鼠股骨和胫骨骨干皮质骨以及6周龄大鼠股骨和胫骨外侧皮质直径2.4毫米缺损处1周龄的修复骨中进行。用于免疫化学研究的骨组织在1%戊二醛、4%多聚甲醛和0.1M磷酸盐缓冲液（pH 7.4）中固定2天。脱钙在6%乙二胺四乙酸中进行2 - 3天。渗透采用Lowicryl树脂K4M，包埋和固化过程在温度为 - 30摄氏度的低温恒温器中进行。使用抗波形蛋白单克隆抗体，采用包埋后技术进行标记。有效的抗体稀释度范围为1:10至1:200，其中1:25和1:100的稀释度显示细丝标记与最少的基质背景的最佳组合。将网格暴露于10纳米金胶体偶联的山羊抗小鼠IgM以显示结合情况。
波形蛋白免疫标记在成骨细胞和骨细胞胞体细胞质内的细丝、成骨细胞和骨细胞整个细胞突起长度上都清晰可见，并且与细胞突起内靠近细胞体处以及远离细胞体的小管内存在的细胞内间隙连接密切相关。
免疫金标记证实大鼠骨的成骨细胞和骨细胞的细胞体及突起中存在中间丝波形蛋白。波形蛋白分布并不集中于特定区域，在细胞体和突起的整个范围内都存在，并且紧邻间隙连接可见。
