September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Chitosan membrane-cultivated keratocyte spheres suppress stromal fibrosis via inhibition of epithelial mesenchymal transition (EMT)
Author Affiliations & Notes
  • Chi-Chin Sun
    Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Keelung, Taiwan
    Department of Chinese Medicine , School of Medicine, Chang Gung University, Taoyuan, Taiwan
  • Kuo-Ying Lee
    Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Keelung, Taiwan
  • Jong-Hwei Su Pang
    Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan
  • Footnotes
    Commercial Relationships   Chi-Chin Sun, None; Kuo-Ying Lee, None; Jong-Hwei Pang, None
  • Footnotes
    Support  NSC99-2314-B-182A-025-MY3
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 2364. doi:
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    • Get Citation

      Chi-Chin Sun, Kuo-Ying Lee, Jong-Hwei Su Pang; Chitosan membrane-cultivated keratocyte spheres suppress stromal fibrosis via inhibition of epithelial mesenchymal transition (EMT). Invest. Ophthalmol. Vis. Sci. 2016;57(12):2364.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Previously, we demonstrated the anti-fibrotic effects of keratocyte spheres cultivated on chitosan membrane. The purpose of this study is to investigate the underlying mechanisms.

Methods : Human corneas stripped of both endothelium and epithelium were placed in a solution of collagenase overnight at 37°C. Confluent cell layers were treated with trypsin-EDTA and seeded onto the chitosan coatings or plastic dishes. We used Affymetrix oligonucleotide microarrays to analyze common transcriptomes and thereby learn about the core gene expression profile in human keratocytes cultured on chitosan membrane on plastic dishes. The advantage of microarray analysis of gene expression is that it can be used to disclose associating genes that were previously thought to be unrelated to a physiological or pathological event. However, interpreting complex biological processes from gene expression profiles often requires extensive knowledge mining in biomedical literature. Therefore, we adopted a commercially available biological database and software program, MetaCore (GeneGo Inc.), for functional network analysisto reveal the common functions of the interacting genes. Real-time PCR, western blotting analysis and immunofluorescence staining were performed to validate the expressions of these genes at RNA and protein levels.

Results : Analysis of gene expression by microarray and MetaCore pathway mapping revealed that regulation of epithelial-to-mesenchymal transition (EMT) was the most important pathway involved in keratocyte spheres cultured on chitosan membrane as compared with on plastic dishes (4.193E-12). Transforming growth factor beta receptor type 1 was significantly down-regulated in chitosan-cultivated keratocyte spheres. Cell motility-associated genes including caldesmon, tropomyosin-1, ACTA2 and ACTB were all down-regulated in keratocyte spheres. Real-time PCR, western blotting analysis and immunofluorescence staining confirmed the findings observed at transcriptional level.

Conclusions : Our studies demonstrated that chitosan membrane-cultivated keratocyte spheres repaired corneal scarring through inhibition of transforming growth factor beta-induced epithelial mesenchymal transition (EMT).

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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