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Effects of 17β-Estradiol on Human Corneal Wound Healing In Vitro

Oh, Tae-Hoon MD; Chang, Dong-Jin MD; Choi, Jun-Sub PhD; Joo, Choun-Ki MD, PhD

doi: 10.1097/ICO.0b013e31823d03ca
Basic Investigation

Purpose: To investigate the effect of 17β-estradiol on corneal wound healing, particularly on epithelial mitosis and migration.

Methods: Immortalized human corneal epithelial cells (HCECs) were cultured in media with different concentrations of 17β-estradiol (10, 50, 100, and 200 pg/mL), Dulbecco modified Eagle medium: Nutrient Mixture F-12 (negative control), and serum-containing Dulbecco modified Eagle medium: Nutrient Mixture F-12 (positive control). After 6 or 24 hours of hormone treatment, to evaluate the migratory potential of 17β-estradiol, wound healing assays were conducted via the manual scraping of HCECs and western blot analysis of fibronectin and matrix metalloproteinase 9 (MMP9). The proliferative potential of 17β-estradiol was evaluated via a proliferation assay using western blot analysis for proliferating cell nuclear antigen. In addition, epidermal growth factor (EGF) was measured by reverse transcription–polymerase chain reaction, and for the inhibition of epidermal growth factor receptor (EGFR)–mediated signal transduction, a wound healing assay was conducted after HCECs cultured with EGFR small interfering RNA were stimulated with 100 pg/mL 17β-estradiol.

Results: Wound healing assay rates were enhanced as 17β-estradiol increased, with statistically significant changes seen in 50, 100, and 200 pg/mL 17β-estradiol–treated and positive control cells, compared with negative control cells (P < 0.05, in each group). Western blot analysis revealed that the expression of the MMP9 gene was upregulated by 17β-estradiol, and the expression of the fibronectin gene was downregulated by 17β-estradiol. The mitosis assay via western blot analysis showed that the expression cell cycle–associated protein, proliferating cell nuclear antigen, increased gradually as a result of 17β-estradiol treatment. Reverse transcription–polymerase chain reaction showed that EGF was upregulated by 17β-estradiol, and the EGFR small interfering RNA did not totally block the wound healing of the 17β-estradiol–treated cells but statistically significantly reduced the wound healing rate (P = 0.031).

Conclusions: 17β-Estradiol facilitated the maintenance of the beneficial effect on corneal epithelial migration and proliferation, and the promoting effect of 17β-estradiol is partially related to increased EGF in vitro.

Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea

Reprints: Choun-Ki Joo, Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, #505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea (e-mail: ckjoo@catholic.ac.kr).

The authors have no funding or conflicts of interest to disclose.

Received August 8, 2010

Accepted July 18, 2011

© 2012 Lippincott Williams & Wilkins, Inc.