Burmeister J S, Truskey G A, Yarbrough J L, Reichert W M
Department of Biomedical Engineering, Duke University, Durham, North Carolina 27706.
Biotechnol Prog. 1994 Jan-Feb;10(1):26-31. doi: 10.1021/bp00025a003.
A simplified model of total internal reflection fluorescence (TIRF) emission of fluorescently labeled cell membranes [Reichert, W. M.; Truskey, G. A. J. Cell Sci. 1990, 96, 219-230] was used to determine the topography of the cell membrane in apposition to a polymer-coated surface. The homopolymer substrates were spun cast films of hydrophilic poly(hydroxyethyl methacrylate) (polyHEMA) or hydrophobic poly(ethyl methacrylate) (polyEMA). Bovine aortic endothelial cells (BAEC) on preadsorbed fibronectin polymer substrates were either plated for 24 h, fixed, labeled, and examined by TIRF microscopy (TIRFM) and phase-contrast microscopy or plated for 2 h and tested for their adhesion strength in a parallel-plate flow chamber. BAEC attached to polyHEMA showed no evidence of focal contact formation. However, BAEC attached to polyEMA were well spread and showed an array of focal contacts. TIRFM data were transformed to construct a detailed topographical map of relative cell/substrate separation distances. Virtually all of the BAEC plated to polyHEMA were sheared from the surface when subjected to a 50 dyn/cm2 burst of laminar flow, whereas only 10% of the BAEC were sheared from the polyEMA surface. These data suggest that the polyHEMA and polyEMA surface properties (e.g., hydrophobicity) correlate with the presence of BAEC focal contacts and the BAEC attachment strength.
采用一种简化的荧光标记细胞膜全内反射荧光（TIRF）发射模型[赖歇特，W.M.；特鲁斯基，G.A.《细胞科学杂志》1990年，96卷，219 - 230页]来确定与聚合物涂层表面相对的细胞膜形貌。均聚物底物是亲水性聚甲基丙烯酸羟乙酯（聚HEMA）或疏水性聚甲基丙烯酸乙酯（聚EMA）的旋涂膜。预吸附纤连蛋白聚合物底物上的牛主动脉内皮细胞（BAEC）要么接种24小时，固定、标记后通过全内反射荧光显微镜（TIRFM）和相差显微镜检查，要么接种2小时并在平行板流动腔中测试其黏附强度。附着在聚HEMA上的BAEC没有形成粘着斑的迹象。然而，附着在聚EMA上的BAEC铺展良好并显示出一系列粘着斑。对TIRFM数据进行转换以构建细胞/底物相对分离距离的详细地形图。当受到50达因/平方厘米的层流冲击时，几乎所有接种到聚HEMA上的BAEC都从表面被冲走，而只有10%的BAEC从聚EMA表面被冲走。这些数据表明聚HEMA和聚EMA表面性质（如疏水性）与BAEC粘着斑的存在以及BAEC附着强度相关。
