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Eun-Soon Kim, Sun Young Kim, Mi Hyun Cheon, Dong-Ah Ko, Jae-hyung Kim, Jae Yong Kim, Myoung Joon Kim, Hungwon Tchah; Expression Of Inflammatory Cytokines And Receptors Using An Ex Vivo, Dry-Eye Model Under Hyperosmotic Stress. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1132.
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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.
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.
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.
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.
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