Vande Berg J S, Rudolph R, Poolman W L, Disharoon D R
San Diego Veterans Administration Medical Center, Scripps Clinic, California.
Lab Invest. 1989 Nov;61(5):532-8.
The normal contraction of open wounds and many forms of pathologic contracture are related by the presence of a contractile fibroblast known as a myofibroblast. The function of this cell has been postulated as a result of previous pharmacological, immunological, and biochemical testing on strips of contracted connective tissue. The purpose of this study was to develop a specific assay that could measure the concentration of one contractile element (actin) within cultured myofibroblasts isolated from a contracting wound and in normal fibroblasts from uninjured dermis. Rates of growth and actin concentration through 15 days of culture were compared among populations of paired control fibroblasts from normal dermis and granulating wound myofibroblasts from three patients. Growth curves showed that myofibroblasts always grew slower than fibroblasts. An enzyme-linked immunosorbent assay showed that actin concentration was generally greater in mass cultures of granulating wound myofibroblasts than in fibroblasts from uninjured dermis. During exponential growth (1-6 days) the average actin concentration among myofibroblast lines ranged from 24 to 62 pg/cell. Average actin levels among control fibroblasts ranged from 3 to 47 pg/cell during the same interval. After 15 days of culture, actin concentration peaked twice. The first actin peak occurred within the period of exponential growth. At confluency, cellular actin levels dropped. Superconfluent cultures exhibited a second actin peak that displayed an irregular pattern of actin concentration. The latter observation suggested an artifact that might be the result of three-dimensional matrix of cells that altered points of cell adhesion and produced an irregular pattern of actin concentration. These data show that the phenotype of increased actin in cultured myofibroblasts was carried over by myofibroblasts from contracted skin wounds to culture. Because of a higher concentration of actin in myofibroblasts than in undifferentiated fibroblasts, these data suggest that the differentiation process of myofibroblasts may be associated with an increased availability of monomeric actin for filament synthesis. This study demonstrates that the use of tissue culture and our enzyme-linked immunosorbent assay will be a useful method to study factors affecting myofibroblast phenotypic modulation. Future studies should be directed toward developing procedures for isolation of pure populations of myofibroblasts as well as extracellular matrices that would maintain the morphology of both differentiated myofibroblasts and normal undifferentiated fibroblasts.
开放性伤口的正常收缩以及多种病理性挛缩，都与一种名为肌成纤维细胞的收缩性成纤维细胞的存在有关。这种细胞的功能是根据先前对收缩结缔组织条带进行的药理学、免疫学和生化测试推测出来的。本研究的目的是开发一种特定的检测方法，该方法能够测量从收缩伤口分离出的培养肌成纤维细胞以及未受伤真皮中的正常成纤维细胞内一种收缩元件（肌动蛋白）的浓度。比较了来自正常真皮的配对对照成纤维细胞群体以及三名患者肉芽伤口肌成纤维细胞群体在15天培养过程中的生长速率和肌动蛋白浓度。生长曲线表明，肌成纤维细胞的生长总是比成纤维细胞慢。酶联免疫吸附测定表明，肉芽伤口肌成纤维细胞的大量培养物中肌动蛋白浓度通常高于未受伤真皮中的成纤维细胞。在指数生长期（1 - 6天），肌成纤维细胞系中的平均肌动蛋白浓度范围为24至62 pg/细胞。在相同时间段内，对照成纤维细胞中的平均肌动蛋白水平范围为3至47 pg/细胞。培养15天后肌动蛋白浓度出现两次峰值。第一个肌动蛋白峰值出现在指数生长期。细胞汇合时，细胞内肌动蛋白水平下降。过度汇合培养物呈现出第二个肌动蛋白峰值，该峰值显示出不规则的肌动蛋白浓度模式。后一观察结果表明这可能是一种假象，可能是由于细胞的三维基质改变了细胞黏附点并产生了不规则的肌动蛋白浓度模式所致。这些数据表明，培养的肌成纤维细胞中肌动蛋白增加的表型从收缩皮肤伤口的肌成纤维细胞延续到了培养环境中。由于肌成纤维细胞中肌动蛋白浓度高于未分化的成纤维细胞，这些数据表明肌成纤维细胞的分化过程可能与用于细丝合成的单体肌动蛋白可用性增加有关。本研究表明，组织培养和我们的酶联免疫吸附测定的应用将是研究影响肌成纤维细胞表型调节因素的一种有用方法。未来的研究应致力于开发分离纯肌成纤维细胞群体以及能维持分化的肌成纤维细胞和正常未分化成纤维细胞形态的细胞外基质的方法。
