Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque
Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque
复制标题
DOI：
10.1007/s004419900074
复制
发表时间：
2000-01-01
影响因子：
3.6
通讯作者：
Murphy, CJ
中科院分区：
文献类型：
--
作者：
Abrams, GA;Goodman, SL;Murphy, CJ
本文定量定义了猕猴角膜前上皮基底膜的纳米级形貌。将从猕猴切下的角膜纽扣置于2.5 mM乙二胺四乙酸盐（EDTA）中2.5 h，之后小心地去除上皮以暴露下面的基底膜。使用荧光显微镜结合针对层粘连蛋白和IV型胶原蛋白的抗体染色以及透射电子显微镜验证剩余基底膜的完整性。基底膜的表面的表征进行了使用透射电子显微镜，高分辨率，低电压扫描电子显微镜，和原子力显微镜。所有三种成像技术获得的定量数据进行比较。基底膜具有复杂的形貌，由紧密交联的纤维与孔混合组成。通过透射电子显微镜、扫描电子显微镜和原子力显微镜测量的特征的平均升高分别为149+/-60 nm、191+/-72 nm和147+/-73 nm。通过SEM测量的平均纤维直径为77+/-44nm，孔径为72+/-40nm，孔占据总表面积的约15%。对于Matrigel（一种市售的基底膜样复合物）也发现了类似的特征类型和尺寸，这支持了通过角膜制备程序去除上覆上皮而引入的伪影最少。地形特征放大了细胞-基质相互作用发生的表面积，估计为400%。基底膜的三维结构表现出丰富的复杂地形的个人功能，包括孔隙和纤维的尺寸范围从30到400 nm。这些纳米级基质特征可以调节基本细胞行为，例如粘附、迁移、增殖和分化。
This paper quantitatively defines the nanoscale topography of the basement membrane underlying the anterior corneal epithelium of the macaque. Excised corneal buttons from macaques were placed in 2.5 mM ethylenediaminetetraacetate (EDTA) for 2.5 h, after which the epithelium was carefully removed to expose the underlying basement membrane. The integrity of the remaining basement membrane was verified using fluorescent microscopy in conjunction with antibody staining directed against laminin and collagen type IV as well as transmission electron microscopy. Characterization of the surface of the basement membrane was performed using transmission electron microscopy, high-resolution, low-voltage scanning electron microscopy, and atomic force microscopy. Quantitative data were obtained with all three imaging techniques and compared. The basement membrane has a complex topography consisting of tightly cross-linked fibers intermingled with pores. The mean elevation of features measured by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy was 149+/-60 nm, 191+/-72 nm, and 147+/-73 nm, respectively. Mean fiber diameter as measured by SEM was 77+/-44 nm and pore diameter was 72+/-40 nm, with pores occupying approximately 15% of the total surface area. Similar feature types and dimensions were also found for Matrigel, a commercially available basement membrane-like complex, supporting that a minimum of artifact was introduced by corneal preparative procedures to remove the overlying epithelium. Topographic features amplified the surface area over which cell-substratum interactions occur by an estimated 400%. The three-dimensional structure of the basement membrane exhibits a rich complex topography of individual features, consisting of pores and fibers with dimensions ranging from 30 to 400 nm. These nanoscale substratum features may modulate fundamental cell behaviors such as adhesion, migration, proliferation, and differentiation.