Immunodetection of connexins and cadherins in corneal fibroblasts and myofibroblasts.
Immunodetection of connexins and cadherins in corneal fibroblasts and myofibroblasts.
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发表时间：
1996-08
影响因子：
4.4
通讯作者：
S. Petridou;S. Masur
中科院分区：
文献类型：
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作者：
S. Petridou;S. Masur
在正常角膜中，基质成纤维细胞（角膜细胞）通过缝隙连接相互作用。角膜创伤后，剩余的角膜基质细胞表型为成纤维细胞和肌成纤维细胞。为了深入了解成纤维细胞和肌成纤维细胞在伤口愈合中的各自作用，作者研究了角膜成纤维细胞和角膜肌成纤维细胞培养物中细胞-细胞相互作用的分子基础。方法采用免疫印迹分析和免疫荧光显微镜，作者确定了连接蛋白连接蛋白，钙粘蛋白和钙粘蛋白相关蛋白（连环蛋白）的相对表达和定位在培养的成纤维细胞和肌成纤维细胞。结果在培养的角膜成纤维细胞中，差距连接蛋白connexin 43高表达并定位于致密斑;在细胞间接触中未检测到钙粘蛋白。培养的肌成纤维细胞表现出相反的模式：钙粘蛋白高度表达和定位在细胞-细胞接触，而肌成纤维细胞连接蛋白43主要是细胞内。肌成纤维细胞钙粘蛋白通过泛钙粘蛋白抗体鉴定为与N-钙粘蛋白单克隆抗体反应弱的135 kDa分子。此外，钙粘蛋白相关的细胞质蛋白，α-和β-连环蛋白，与钙粘蛋白共定位在肌成纤维细胞的细胞-细胞边界。结论：角膜成纤维细胞细胞间连接蛋白43的存在与融合角膜成纤维细胞中连接的主要通讯作用一致。相反，在肌成纤维细胞的细胞-细胞边界处钙粘蛋白的存在可能为肌动蛋白丝的插入提供位点。钙粘蛋白-肌动蛋白协会可以支持肌动蛋白为基础的力量产生有效的伤口闭合。
PURPOSE In normal cornea, stromal fibroblasts (keratocytes) interact with one another by gap junctions. After corneal wounding, the remaining corneal stroma cells are phenotypically fibroblasts and myofibroblasts. For insight into the respective roles of fibroblasts and myofibroblasts in wound healing, the authors have investigated the molecular basis of cell-cell interaction in cultures of corneal fibroblasts and corneal myofibroblasts. METHODS Using Western blot analysis and immunofluorescent microscopy, the authors determined the relative expression and localization of junction proteins-connexins, cadherins, and cadherin-associated proteins (catenins)-in cultured fibroblasts and myofibroblasts. RESULTS In cultured corneal fibroblasts, the gap junction protein, connexin 43, was highly expressed and was localized to dense maculae; cadherins were not detected in cell-cell contacts. Cultured myofibroblasts showed the opposite pattern: Cadherins were highly expressed and localized at the cell-cell contacts, whereas myofibroblast connexin 43 was primarily intracellular. Myofibroblast cadherin was identified by a pan-cadherin antibody as a molecule of 135 kDa that reacted weakly with an N-cadherin monoclonal antibody. In addition, cadherin-associated cytoplasmic proteins, alpha- and beta-catenins, co-localized with cadherin at the cell-cell borders of the myofibroblasts. CONCLUSIONS The presence of connexin 43 at the cell-cell borders of corneal fibroblasts is consistent with a primary communication role of junctions in confluent corneal fibroblasts. In contrast, the presence of cadherin at the cell-cel borders of myofibroblasts may provide a site for insertion of actin filaments. A cadherin-actin association could support actin-based force generation for effective wound closure.