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R. Yoshioka, A. Shiraishi, T. Kobayashi, S.-I. Morita, Y. Hayashi, S. Higashiyama, Y. Ohashi; Role of HB-EGF in Corneal Epithelial Wound Healing in vivo and in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2957. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Heparin binding EGF-like growth factor (HB-EGF) is a member of the EGF family that plays an important role in development and regeneration of various tissues. To investigate the function of HB-EGF in corneal epithelial wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter; HBlox/lox: K5-Cre (HB-/-). To determine the precise mechanisms of HB-EGF, we employed cultured corneal epithelial cells isolated from HB-/- (HB-/-CECs).
Epithelial defects (2.0 mm) were created in the center of corneas of 6-8 weeks old HB-/-CECs and HBlox/lox, and the wounded area were monitored up to 5days. Corneal epithelial cells were isolated from HB-/-, and cultured under serum-free conditions. Third- or fourth-passage cells were used in this study. For in vitro wound healing assay, confluent HB-/-CECs were scraped with a 1ml pipette tip, and the extent of remaining acellular area were measured at 0, 12 and 24 hours after scraping in the presence or absence of 10ng/ml of HB-EGF. For cell attachment assay, HB-/-CECs were seeded to 96 well plate in 5×104cells/well, after 2 hour incubation in the presence or absence of HB-EGF, the number of cells adhered to the dishes were counted.
In vivo corneal wound was healed at 48hr in HBlox/lox, but it takes 86.7hr in HB-/-. Addition, the re-detachment of regenerated epithelium was frequently observed in HB-/-. In vitro wound healing, remaining acellular area at 0, 12, 24 hours after wounding was 100%, 77%, 60% in the absence of HB-EGF, 100%, 23%, 0% in the presence of HB-EGF, respectively. For cell attachment assay, 9% of the cells were attached in the absence of HB-EGF, while 16% of the cells were attached in the presense of HB-EGF.
Corneal epithelial wound healing was impaired in HB-EGF deficient mouse corneas in vivo and in vitro.
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