Zieske J D, Mason V S, Wasson M E, Meunier S F, Nolte C J, Fukai N, Olsen B R, Parenteau N L
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114.
Exp Cell Res. 1994 Oct;214(2):621-33. doi: 10.1006/excr.1994.1300.
A three-dimensional corneal tissue construct was used to examine the effect of culture environment and endothelial cell interaction on epithelial differentiation and basement membrane assembly. Rabbit corneal epithelial cells were cultured over rabbit stromal fibroblasts in a collagen matrix with or without an underlying layer of immortalized mouse corneal endothelial cells (Muragaki, Shiota, Inoue, Ooshima, Olsen, and Ninomiya. (1992) Eur. J. Biochem. 207, 895-902). The cultures were grown submerged or at a dry or moist interface. Basement membrane, anchoring fibril, and hemidesmosome assembly was monitored using transmission electron microscopy as well as indirect immunofluorescence microscopy of laminin, type VII collagen, and alpha 6 integrin. Antibodies against keratin 3 (K3) and alpha-enolase marked differentiated and undifferentiated corneal epithelial cells, respectively. When all three cell types were cultured at a moist interface, hemidesmosomes, anchoring fibrils, and a continuous basement membrane were observed 2 wk after lifting the cultures to an air-liquid interface (air-lift). The distribution of alpha-enolase and K3 was identical to patterns seen in the limbal region of the cornea. Air-lifted tissue constructs lacking the endothelial cell layer showed only limited distribution of laminin and type VII collagen at the epithelial-matrix junction. alpha 6 Integrin was present along the entire plasma membrane of the basal cells; epithelial differentiation was not complete as alpha-enolase was seen in basal and two to three layers of suprabasal cells. Submerged cultures without endothelial cells did not express differentiation markers or basement membrane components. These data indicate that endothelial cell interaction dramatically enhances the amount and quality of epithelial basement membrane assembly and that epithelial differentiation is influenced by the type of interface between tissue, liquid, and air.
使用三维角膜组织构建体来研究培养环境和内皮细胞相互作用对上皮分化和基底膜组装的影响。将兔角膜上皮细胞培养在兔基质成纤维细胞上，置于有或没有永生化小鼠角膜内皮细胞底层的胶原基质中（村垣、盐田、井上、大岛、奥尔森和二宫。（1992年）《欧洲生物化学杂志》207卷，895 - 902页）。培养物在浸没状态下生长，或在干燥或湿润界面生长。使用透射电子显微镜以及层粘连蛋白、VII型胶原和α6整合素的间接免疫荧光显微镜监测基底膜、锚定纤维和半桥粒的组装。抗角蛋白3（K3）抗体和α - 烯醇化酶抗体分别标记分化和未分化的角膜上皮细胞。当所有三种细胞类型在湿润界面培养时，将培养物提升至气液界面（气提）2周后，观察到半桥粒、锚定纤维和连续的基底膜。α - 烯醇化酶和K3的分布与在角膜缘区域观察到的模式相同。缺乏内皮细胞层的气提组织构建体在上皮 - 基质交界处仅显示层粘连蛋白和VII型胶原的有限分布。α6整合素存在于基底细胞的整个质膜上；上皮分化不完全，因为在基底细胞和两到三层基底上层细胞中可见α - 烯醇化酶。没有内皮细胞的浸没培养物不表达分化标志物或基底膜成分。这些数据表明内皮细胞相互作用显著增强上皮基底膜组装的数量和质量，并且上皮分化受组织、液体和空气之间界面类型的影响。
