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Wenjuan Qin, Yangluowa Qu, Stephen C Pflugfelder, De-Quan Li; Limbal Stem Cells Promote Corneal Epithelial Regeneration by GDNF/Survivin/CRM1 Signaling Pathway. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3871. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the novel mechanism by which limbal stem cells promote regeneration of human corneal epithelium by GDNF/Survivin/CRM1 signaling pathway.
Fresh donor corneal tissues were used to make cryosections and to generate primary human corneal epithelial cells (HLECs) in 12-well plates. When the cultures grew to confluence, a 2 mm-wide zone of epithelial cells were scraped to create an in vitro wound model for investigation of the corneal epithelial regeneration in the conditions with or without rhGDNF treatment and in the presence or absence of rhGDNF antibody. The mRNA expression was evaluated by reverse transcription and Real-time PCR, and their proteins were detected by ELISA, immunofluorescent staining and Western blotting.
GDNF and survivin proteins were found to be immunolocalized in the basal cells of limbal epithelium. GDNF production was upregulated at mRNA and protein levels in scraping-created wound model of primary HCECs. The corneal cells could spontaneously proliferate to heal the 2 mm-wide wound within 48 hours, and exogenous rhGDNF promoted the healing completed as fast as in 24 hours. Neutralizing GDNF antibody markedly inhibited the epithelial regeneration to heal the wound in either condition without or with rhGDNF addition. Interestingly, the mRNA expression of survivin were significantly upregulated in 4-24 hours after wounding, accompanied by activation of survivin proteins, evidenced by nuclear translocation of their immunoreactivities from the cytoplasm. Furthermore, the nuclear translocation of survivin activated by GDNF was concomitant with the downregulated expression and deactivated protein of CRM1/nuclear exportin-1, which was translocated from nucleus to cytoplasm at the wounding edge after 4-8 hours.
These findings demonstrate that limbal stem cells promote regeneration of human corneal epithelium by producing GDNF via a signaling pathway that activates survivin while deactivates CRM1.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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