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R. Yoshioka, A. Shiraishi, T. Kobayashi, E. Noda, Y. Hayashi, Y. Ohashi; HB-EGF is Essential for Promotion of Cell Attachment and Proliferation in Corneal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2010;51(13):384.
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We previously reported that keratinocyte-specific HB-EGF-deficient mice (HB-/-) showed impaired corneal epithelial healing with frequent detachment of the regenerated epithelium. In this study, we attempted to determine the role of HB-EGF in corneal wound healing in vitro using cultured corneal epithelial cells of HB-/- (HB-/- MCECs) and wild type mice (WT MCECs).
Primary culture of HB-/- and WT corneal epithelial cells was carried out under serum-free conditions. Confluently grown MCECs of each type were scraped with a 1 ml pipette tip, and wound closure in the presence or absence of 10 ng/ml HB-EGF was comparatively monitored at 0, 12 and 24 hours after scraping. Cell proliferation was assessed by examining BrdU incorporation after 24 hours. The expression of EGF family mRNA was evaluated by real-time PCR at 0.5, 1, 3, 6, and 24 hours after scraping. In the cell attachment assay, MCECs were pre-incubated either with or without HB-EGF for 24 hours, and seeded onto 96 well plates at a concentration of 5×104cells/well. After incubation for 2 hours, the number of cells adhering to the dishes was counted.
Wound closure was significantly impaired in HB-/- MCECs, although similar numbers of BrdU positive cells were observed in the WT (31.6 cells/1000cells) and HB-/- samples (26.6 cells/1000cells). When HB-EGF was added, wound closure accelerated, and increased numbers of BrdU positive cells were found in both the WT (83.2 cells/1000cells) and HB-/- samples (49.7 cells/1000cells). The cell adhesion rate of HB-/- MCECs (12.7%) was lower than that of WT MCECs (21.8%), but increased when HB-EGF was added. Epiregulin mRNA expression was found to be up-regulated in HB-/- MCECs in both the absence and presence of HB-EGF.
HB-EGF was shown to be essential for uneventful corneal epithelial healing through promotion of cell attachment and proliferation, partly with the aid of epiregulin.
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