Pereira Renata O, Carvalho Simone N, Stumbo Ana Carolina, Rodrigues Carlos A B, Porto Luis Critóvão, Moura Anibal S, Carvalho Laís
Laboratório de Cultura de Células, Departamento de Histologia e Embriologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
In Vitro Cell Dev Biol Anim. 2006 Jan-Feb;42(1-2):4-7. doi: 10.1007/s11626-006-0003-0.
Skeletal fibroblasts in vitro can acquire myofibroblast phenotypes by the development of biochemical and morphological features, mainly the expression of alpha-smooth-muscle actin (alpha-SMA). Myogenic differentiation is a central event in skeletal muscle development, and has commonly been studied in vitro in the context of skeletal muscle development and regeneration. Controlling this process is a complex set of interactions between myoblasts and the extracellular matrix. Osteopontin (OPN) is an acidic, phosphorylated matrix protein that contains an Arg-Gly-Asp (RGD) cell attachment sequence and has been identified as an adhesive and migratory substrate for several cell types. The aim of this study was to investigate osteopontin expression during the differentiation of skeletal fibroblasts into myofibroblasts and during myogenesis in a coculture model. Fibroblasts and myoblasts were obtained from skeletal muscle of 18-d-old Wistar strain rat fetuses by enzymatic dissociation. At 1 and 9 d, cocultures were immunolabeled, and the cells were also separately subjected to Western blotting to analyze OPN expression. Our data using confocal microscopy showed that myoblasts displayed a strong staining for OPN and that this labeling was maintained after myotube differentiation. Conversely, during fibroblast differentiation into myofibroblasts, we observed a significant increase in OPN expression. The results obtained by immunolabeling were confirmed by Western blotting. We suggest that OPN is important mainly during early stages of myogenesis, facilitating myoblast fusion and differentiation, and that the increased expression of OPN in myofibroblasts might be related to its effects as a key cytokine regulating tissue repair and inflammation.
体外培养的骨骼成纤维细胞可通过生化和形态学特征的发展获得肌成纤维细胞表型，主要是α-平滑肌肌动蛋白（α-SMA）的表达。肌源性分化是骨骼肌发育中的核心事件，并且通常在骨骼肌发育和再生的背景下进行体外研究。控制这一过程是成肌细胞与细胞外基质之间一系列复杂的相互作用。骨桥蛋白（OPN）是一种酸性磷酸化基质蛋白，含有精氨酸-甘氨酸-天冬氨酸（RGD）细胞附着序列，已被确定为几种细胞类型的粘附和迁移底物。本研究的目的是在共培养模型中研究骨桥蛋白在骨骼成纤维细胞向肌成纤维细胞分化过程以及肌生成过程中的表达情况。通过酶解从18日龄Wistar品系大鼠胎儿的骨骼肌中获取成纤维细胞和成肌细胞。在第1天和第9天，对共培养物进行免疫标记，并且还分别对细胞进行蛋白质印迹分析以检测OPN表达。我们使用共聚焦显微镜得到的数据显示，成肌细胞对OPN呈现强染色，并且这种标记在肌管分化后得以维持。相反，在成纤维细胞向肌成纤维细胞分化过程中，我们观察到OPN表达显著增加。免疫标记获得的结果通过蛋白质印迹得到了证实。我们认为，OPN主要在肌生成的早期阶段发挥重要作用，促进成肌细胞融合和分化，并且肌成纤维细胞中OPN表达的增加可能与其作为调节组织修复和炎症的关键细胞因子的作用有关。
