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Marie A. Shatos, José D. Ríos, Yoshitaka Horikawa, Robin R. Hodges, Eli L. Chang, Carlo R. Bernardino, Peter A. D. Rubin, Darlene A. Dartt; Isolation and Characterization of Cultured Human Conjunctival Goblet Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(6):2477-2486. doi: 10.1167/iovs.02-0550.
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purpose. To isolate and characterize goblet cells from normal human conjunctival tissue to determine whether epidermal growth factor (EGF) receptors are present and whether EGF can influence goblet cell proliferation.
methods. Goblet cells were isolated from explant cultures established from normal conjunctival tissue harvested from patients during periocular surgery. The cells were grown in RPMI culture medium supplemented with 10% fetal bovine serum and characterized using morphology, histochemistry, indirect immunofluorescence microscopy, molecular biology, and biochemistry. Proliferation was determined with a MTT proliferation assay after exposing goblet cells, which had been serum deprived for 48 hours, to increasing concentrations of epidermal growth factor (EGF; 0–80 ng/mL) for 24 hours.
results. Goblet cells were isolated from conjunctival explants by scraping nongoblet cells from the culture dish. Human goblet cells exhibited positive reactivity with alcian blue-periodic acid Schiff (PAS) reagent, goblet cell-specific cytokeratin-7, HPA lectin, and MUC5AC, but negative reactivity to the stratified squamous epithelial cell marker, cytokeratin-4. The mRNA for MUC5AC was detected using RT-PCR. The presence of the EGF receptors EGFR, ErbB2, and ErbB3 was confirmed through Western blot analysis of cell lysates. EGF elicited a concentration-dependent increase in goblet cell proliferation of 160% ± 0.5%, 188% ± 0.45%, 293% ± 1.3%, and 220% ± 0.5% of control values with 10, 20, 40, and 80 ng/mL EGF, respectively.
conclusions. Human goblet cells that retain characteristics of goblet cells in vivo can be cultured. EGF receptors are present in human goblet cells, and EGF stimulates their proliferation.
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