PURPOSE: This study was designed to determine at the molecular level whether interactions between the opioid growth factor (OGF) and OGF receptor (OGFr) play a role in regulating DNA synthesis in the homeostasis of the corneal epithelium. METHODS: The plasmid pcDNA3.1+OGFr-HA, carrying the rat OGFr cDNA epitope-tagged with a C-terminal hemagglutinin (HA), or the empty-vector (pcDNA3.1+), was delivered twice by the Helios Gene Gun System at 300 psi to the cornea of anesthetized rats. The contralateral (untreated) cornea served as the naive specimen. BrdU was used to determine whether the recombinant OGFr was effective in regulating DNA synthesis in the rat peripheral corneal epithelium. RESULTS: Within 18 hours of transfection, positive HA staining was apparent in both the basal and suprabasal layers (efficiency > 90% of the cells) throughout the central and peripheral cornea. Quantitative immunohistochemistry with rhodamine-conjugated anti-OGFr antibodies revealed twofold more OGFr expression in the central and peripheral epithelium of transfected corneas relative to naive corneas. The number of BrdU-positive basal cells in the peripheral epithelium of the transfected cornea was one-third of that in the naive cornea. CONCLUSIONS: These data demonstrate the direct role of the OGF-OGFr system in determining cellular renewal in the mammalian corneal epithelium. Moreover, the successful establishment of a novel delivery system of cDNAs to the ocular surface suggests a therapeutic role for gene therapy in the eye.
PURPOSE: This study was designed to determine at the molecular level whether interactions between the opioid growth factor (OGF) and OGF receptor (OGFr) play a role in regulating DNA synthesis in the homeostasis of the corneal epithelium. METHODS: The plasmid pcDNA3.1+OGFr-HA, carrying the ratOGFr cDNA epitope-tagged with a C-terminal hemagglutinin (HA), or the empty-vector (pcDNA3.1+), was delivered twice by the Helios Gene Gun System at 300 psi to the cornea of anesthetized rats. The contralateral (untreated) cornea served as the naive specimen. BrdU was used to determine whether the recombinant OGFr was effective in regulating DNA synthesis in the rat peripheral corneal epithelium. RESULTS: Within 18 hours of transfection, positive HA staining was apparent in both the basal and suprabasal layers (efficiency > 90% of the cells) throughout the central and peripheral cornea. Quantitative immunohistochemistry with rhodamine-conjugated anti-OGFr antibodies revealed twofold more OGFr expression in the central and peripheral epithelium of transfected corneas relative to naive corneas. The number of BrdU-positive basal cells in the peripheral epithelium of the transfected cornea was one-third of that in the naive cornea. CONCLUSIONS: These data demonstrate the direct role of the OGF-OGFr system in determining cellular renewal in the mammalian corneal epithelium. Moreover, the successful establishment of a novel delivery system of cDNAs to the ocular surface suggests a therapeutic role for gene therapy in the eye.
Authors: Lauro Augusto de Oliveira; Charles Kim; Luciene Barbosa de Sousa; Ivan R Schwab; Mark I Rosenblatt Journal: Arq Bras Oftalmol Date: 2010 Sep-Oct Impact factor: 0.872