Induction of apoptosis in human corneal epithelial cells in vitro.

摘要

PURPOSE: The purpose of this study was to evaluate exhausted-medium-induced apoptosis in human corneal epithelial (HCE) cells in vitro. METHODS: Confluent HCE cells were maintained in tissue-culture medium for 5-7days. The supernatant was harvested and fresh HCE cells were exposed to the exhausted supernatant for 24-48 h over a range of pH conditions. The exhausted medium was assayed for cytokines interleukin 6 (IL-6), IL-8, interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) by ELISA. Apoptotic cells were assessed using Hoechst 33342 Propidium iodide and AnnexinV-FITC staining and by the TUNEL assay. RESULTS: Human corneal epithelial (HCE) cells exposed to exhausted medium demonstrated a high incidence of apoptosis, which increased over time to 87+/-5% after 48 h. Apoptosis was independent of pH. Cytokine levels were not elevated in the exhausted medium. CONCLUSIONS: This study suggests that exhausted medium can be used as a simple, rapid and potent positive control for apoptosis of HCE cells.