Nishimura T, Toda S, Mitsumoto T, Oono S, Sugihara H
Department of Ophthalmology, Saga Medical School, Saga, Japan.
Exp Eye Res. 1998 Jan;66(1):105-16. doi: 10.1006/exer.1997.0419.
In the cornea, corneal epithelial cells are in close contact with keratocytes: the epithelial cells organize thickened lamellar structure on a layer of keratocytes embedded in extracellular matrix (ECM). Thus, growth factors are expected to critically regulate corneal component cells under epithelial-keratocyte interaction. The purpose of this study is to clarify effects of hepatocyte growth factor (HGF), transforming growth factor-beta1 (TGF-beta1) or epidermal growth factor (EGF) on corneal epithelial cells under epithelial-keratocyte interaction. We examined proliferation and differentiation of the epithelial cells in a simple corneal reconstruction culture composed of an epithelial cell layer on the keratocyte-containing stromal layer, using three-dimensional collagen gel matrix culture. We observed the morphological change by phase contrast microscopy, and conducted histological and immunohistochemical examinations. The epithelial proliferation was examined by nuclear bromodeoxy-uridine (BrdU) uptake. In the reconstructed cornea under epithelial-keratocyte interaction, EGF-, TGF-beta1- and HGF-treated cells formed a thickened epithelial layer consisting of 5-6, 5-6 and 3-4 cells, respectively. In fact, both EGF and TGF-beta1 induced significantly higher intakes of nuclear BrdU of the epithelial cells than HGF. In lamellar differentiation of the epithelial cells, TGF-beta1- or HGF-treated cells formed a triple lamellar structure specific for the in vivo corneal epithelium: basal, middle and superficial layers are composed of cuboidal basal-like cells, spindle wing-like cells and flat superficial-like cells, respectively. TGF-beta1-treated cells formed a more markedly thickened epithelial layer than HGF-treated cells. In contrast, EGF formed a single lamellar structure consisting of cuboidal cells. These results suggest that those growth factors regulate proliferation and/or lamellar differentiation of corneal epithelial cells under epithelial-keratocyte interaction. The most interesting result was that TGF-beta1 promotes proliferation and lamellar differentiation of corneal epithelial cells through keratocyte-mediated stimulation.
在角膜中，角膜上皮细胞与角膜细胞紧密接触：上皮细胞在嵌入细胞外基质（ECM）的一层角膜细胞上组织形成增厚的层状结构。因此，生长因子有望在上皮-角膜细胞相互作用下严格调节角膜组成细胞。本研究的目的是阐明肝细胞生长因子（HGF）、转化生长因子-β1（TGF-β1）或表皮生长因子（EGF）在上皮-角膜细胞相互作用下对角膜上皮细胞的影响。我们使用三维胶原凝胶基质培养，在由含角膜细胞的基质层上的上皮细胞层组成的简单角膜重建培养物中检查上皮细胞的增殖和分化。我们通过相差显微镜观察形态变化，并进行组织学和免疫组织化学检查。通过核溴脱氧尿苷（BrdU）摄取检查上皮细胞增殖。在上皮-角膜细胞相互作用下的重建角膜中，经EGF、TGF-β1和HGF处理的细胞分别形成了由5-6、5-6和3-4个细胞组成的增厚上皮层。事实上，EGF和TGF-β1诱导的上皮细胞核BrdU摄取均显著高于HGF。在上皮细胞的板层分化中，经TGF-β1或HGF处理的细胞形成了体内角膜上皮特有的三层板层结构：基底层、中间层和表层分别由立方体形基底样细胞、纺锤形翼样细胞和平坦的表层样细胞组成。经TGF-β1处理的细胞形成的上皮层比经HGF处理的细胞更明显增厚。相比之下，EGF形成了由立方体细胞组成的单层板层结构。这些结果表明，这些生长因子在上皮-角膜细胞相互作用下调节角膜上皮细胞的增殖和/或板层分化。最有趣的结果是，TGF-β1通过角膜细胞介导的刺激促进角膜上皮细胞的增殖和板层分化。
