Abstract
Purpose: :
To establish an ex vivo, dry-eye model using a multi-layered culture of human conjunctival epithelial cells (HCECs) under hyperosmotic stress and to investigate the expression of inflammatory cytokines and receptors in the ex vivo, dry-eye model according to the high EGF condition.
Methods: :
HCECs were obtained from brain-dead or expired donors and primary mono-layered culture was performed using bronchial epithelial growth medium(BEGM, Clonetics Corp) for one week. Multi-layered culture using air-liquid interface (ALI) media (1:1 mixture of BEGM and DMEM) was done for two weeks. They were cultured in the same concentrations of all supplements, except for EGF, which was used at 0.5 ng/ml. The ex vivo, dry-eye model was exposed for one or two days under hyperosmotic stress (350~450 mOsm/kg), obtained by adding 5M NaCl. The high EGF condition was created by adding 25 ng/mL of EGF to the ALI media. The expressions of inflammatory cytokines and receptors such as EGF, EGFR, IL-6, IL-8, IL-1β, and VEGF were measured using real time PCR (Roche diagnostics). The concentrations of IL-1β, IL-6, IL-8, TNF-α, and VEGF in the supernatant culture media were analyzed using Bio-Plex cytokine asssays (Bio-Rad Laboratories) after one day's exposure. Apoptosis was analyzed using TUNEL staining.
Results: :
The control group (312 mOsm/kg) and the hyperosmotic group (400 mOsm/kg) were exposed for one day. The mRNA of the EGF, EGFR, IL-6, IL-8, IL-1β, and VEGF in the usual EGF group were increased respectively in the high EGF group. The average concentrations of IL-1β, IL-6, IL-8, TNF-α, and VEGF were measured in the control group and also increased respectively in the high EGF group.
Conclusions: :
The expression of inflammatory cytokines and receptors were increased under hyperosmotic conditions using an ex vivo, dry-eye model. The high EGF might alter the responses of inflammatory cytokines in a condition of severe hyperosmotic stress condition.
Keywords: inflammation • cytokines/chemokines • cornea: tears/tear film/dry eye