R&D Laboratories, POLA PHARMA INC., 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812, Japan.
J Dermatol Sci. 2011 Nov;64(2):119-26. doi: 10.1016/j.jdermsci.2011.08.003. Epub 2011 Aug 22.
Fibroblast activation is strongly influenced by mechanical environment in the wound-healing process, especially in fibrosis. Mechanically stressed three-dimensional collagen embedded culture is a useful model representing fibroblasts in morphological as well as biochemical situations encountered during fibrosis.
To find key proteins involved in reducing the number of fibroblasts during mechanical stress, we performed two-dimensional gel electrophoresis (2DE)-based differential display and siRNA-based functional screening with collagen gel culture focusing on the differences between attached and detached culture environments.
Membrane extracts of fibroblasts from 1 day of attached or detached cultures were subjected to 2DE. We compared protein expression levels and identified the attached-culture-dominant proteins by MALDI-TOF-MS. Next, fibroblasts were transfected with siRNA and embedded in collagen gel. Cell number was counted after 3 days in culture.
Eight attached culture dominant proteins were identified with MALDI-TOF-MS. Transfection of siRNA against these proteins demonstrated that electron transfer flavoprotein β subunit (ETFB)-specific siRNA reduced the cell number in the attached culture without a decrease in the detached culture.
ETFB participates in the mechanoregulation of fibroblast cell number in collagen gel culture.
在伤口愈合过程中，纤维化过程强烈受到细胞外基质机械环境的影响。受机械应力的三维胶原包埋培养是一种有用的模型，它在形态和生物化学方面代表了成纤维细胞在纤维化过程中遇到的情况。
为了找到在机械应激下减少成纤维细胞数量的关键蛋白，我们使用胶原凝胶培养进行了基于二维凝胶电泳（2DE）的差异显示和基于 siRNA 的功能筛选，重点关注附着和分离培养环境之间的差异。
对附着或分离培养 1 天的成纤维细胞膜提取物进行 2DE。我们比较了蛋白质表达水平，并通过 MALDI-TOF-MS 鉴定了附着培养优势蛋白。接下来，将成纤维细胞转染 siRNA 并嵌入胶原凝胶中。培养 3 天后计数细胞数量。
通过 MALDI-TOF-MS 鉴定了 8 种附着培养优势蛋白。针对这些蛋白的 siRNA 转染表明，电子传递黄素蛋白β亚基（ETFB）特异性 siRNA 减少了附着培养中的细胞数量，而在分离培养中没有减少。
ETFB 参与了胶原凝胶培养中成纤维细胞数量的机械调节。
