Vishwanath Mridula, Ma Lisha, Otey Carol A, Jester James V, Petroll W Matthew
Department of Ophthalmology, Southwestern Medical Center, Dallas, Texas 75390-9057, USA.
Invest Ophthalmol Vis Sci. 2003 Nov;44(11):4724-35. doi: 10.1167/iovs.03-0513.
To investigate the migratory and contractile behavior of isolated human corneal fibroblasts in fibrillar collagen matrices.
A telomerase-infected, extended-lifespan human corneal fibroblast cell line (HTK) was transfected by using a vector for enhanced green fluorescent protein (GFP)-alpha-actinin. Cells were plated at low density on top of or within 100-microm-thick fibrillar collagen lattices. After 18 hours to 7 days, time-lapse imaging was performed. At each 1- to 3-minute interval, GFP and Nomarski differential interference contrast (DIC) images were acquired in rapid succession. Serum-containing (S+) medium was used initially for perfusion. After 2 hours, perfusion was switched to either serum-free (S-) or S+ medium containing the Rho-kinase inhibitor Y-27632 for 1 to 2 hours. Finally, perfusion was changed back to S+ medium for 1 hour.
Two to 4 days after plating, many cells underwent spontaneous contraction and/or relaxation in S+ medium. A decrease in the distance between consecutive alpha-actinin-dense bodies along stress fibers was measured during contraction, and focal adhesion and matrix displacements correlated significantly. Removal of serum or inhibition of Rho-kinase induced cell body elongation and relaxation of matrix stress, as confirmed using finite element modeling. Rapid formation and extension of pseudopodia and filopodia were also observed, and transient tractional forces were generated by these extending processes.
Cultured human corneal fibroblasts can undergo rapid changes in the subcellular pattern of force generation that are mediated, in part, by Rho-kinase. Sarcomeric shortening of stress fibers in contracting corneal fibroblasts is also demonstrated for the first time.
研究分离的人角膜成纤维细胞在纤维状胶原基质中的迁移和收缩行为。
使用增强型绿色荧光蛋白（GFP）-α-肌动蛋白载体转染端粒酶感染的、寿命延长的人角膜成纤维细胞系（HTK）。将细胞低密度接种在100微米厚的纤维状胶原晶格顶部或内部。18小时至7天后，进行延时成像。每隔1至3分钟，快速连续采集GFP和诺马斯基微分干涉对比（DIC）图像。最初使用含血清（S+）培养基进行灌注。2小时后，将灌注切换至无血清（S-）或含Rho激酶抑制剂Y-27632的S+培养基中1至2小时。最后，将灌注换回S+培养基1小时。
接种后2至4天，许多细胞在S+培养基中发生自发收缩和/或松弛。收缩过程中，沿着应力纤维的连续α-肌动蛋白密集体之间的距离减小，粘着斑和基质位移显著相关。使用有限元模型证实，去除血清或抑制Rho激酶可诱导细胞体伸长和基质应力松弛。还观察到伪足和丝状伪足的快速形成和延伸，这些延伸过程产生瞬时牵引力。
培养的人角膜成纤维细胞可经历亚细胞力产生模式的快速变化，这部分由Rho激酶介导。首次证明收缩的角膜成纤维细胞中应力纤维的肌节缩短。
