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R. Yoshioka, A. Shiraishi, T. Kobayashi, S.-I. Morita, Y. Hayashi, S. Higashiyama, Y. Ohashi; HB-EGF Accelerates Mouse Corneal Epithelial Cell Migration and Attachment. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6307.
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We reported that impaired corneal wound healing and the re-detachment of regenerated epithelium were frequently observed in keratinocyte-specific HB-EGF-deficient mice (HB-/-). To determine the precise mechanisms of HB-EGF, we employed cultured corneal epithelial cells isolated from HB-/- (HB-/-MCEs) and wild type mice (WT MCEs).
Corneal epithelial cells were isolated from HB-/- and WT mice, and cultured under serum-free conditions. Confluent HB-/-MCEs and WT MCEs were scraped with a 1 ml pipette tip, and the extent of the remaining a-cellular area was measured at 0, 12 and 24 hours after scraping in the presence or absence of 10 ng/ml of HB-EGF. BrdU positive cells were counted 44 hours after scraping. In a cell attachment assay, MCEs, which were pre-incubated either with or without HB-EGF for 24 hours, were seeded onto 96 well plates at a concentration of 5×104cells/well. After incubation for 2 hours, the cells adhering to the dishes were counted.
In the absence of HB-EGF, 12 and 24 hours after wounding, 70% and 47% of the a-cellular area remained in HB-/-MCEs, and 49% and 17% remained in WT MCEs, respectively. In the presence of HB-EGF at the same time points, 8% and 0.2% of the a-cellular area remained in HB-/-MCEs, and 39% and 9% remained in WT MCEs. In HB-/-MCEs, 14.4 and 15.1 BrdU positive cells/field were counted in the absence and presence of HB-EGF, respectively. In the cell attachment assay, 12.7% of the HB-/- cells were attached in the absence of HB-EGF, significantly lower than the rate of WT cells (21.8%), while pre-incubation with HB-EGF increased the rate of cell attachment to 25.1% for HB-/- and 26.4% for WT.
HB-EGF may accelerate epithelial cell migration and attachment in corneal epithelial wound healing in mice.
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