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A. Shiraishi, T. Kobayashi, L. Yang, Y. Shirakata, K. Hashimoto, Y. Ohashi; The Expression Pattern of Growth Factors in Fibroblasts in the Collagen Gel Layer of a Three Dimensional Culture of Corneal Epithelial Cell Sheets. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4546.
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© ARVO (1962-2015); The Authors (2016-present)
Epithelial-stromal interaction has been thought to play a role in maintaining corneal epithelial cell migration, proliferation and differentiation. In this study, the expression kinetics of growth factors in fibroblasts in the collagen gel layer of a three dimensional culture of corneal epithelial cell sheets were examined.
Collagen gels (24 mm in diameter) were prepared with human dermal fibroblasts (HDFs, 5x105). Rabbit corneal epithelial cells (RCEs, 2nd pass, 5x105) were seeded onto the denuded amnion attached to the collagen gel. After reaching confluency, the cells were exposed to an air-liquid interface (AL) for 7 days. Total RNA was extracted from fibroblasts in the collagen gel before seeding, and 1, 3, and 7 days after initial exposure to the AL. Using real-time PCR, the expression of growth factors (KGF, HGF, NGF, SCF, EGF, TGF, TGFβ-1, 2, 3) in the RCE-seeded fibroblasts (RCE(+)) was compared with that of fibroblasts in collagen gel without RCEs (RCEs(-)).
RCEs were stratified into 2-3 layers after 3 days of AL exposure and were multilayered after 7 days of AL exposure. After AL exposure, the expression of KGF and TGF was greater in RCEs(+) compared with RCEs(-), while SCF and TGFβ-2 were expressed more strongly in RCEs(-) than in RCEs(+). The expression pattern of TGF was noteworthy, as expression was detected in RCEs(+) after AL exposure, but little or no expression was found in RCEs(-).
The difference in the expression patterns of growth factors in RCEs(+) and RCEs(-) suggest the possibility that growth factors expressed by fibroblasts in collagen gel are influenced by corneal epithelial cells.
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