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Patric Turowski, Peter Adamson, Julie Sathia, Jin Jun Zhang, Stephen E. Moss, G. William Aylward, Matthew J. Hayes, Naheed Kanuga, John Greenwood; Basement Membrane-Dependent Modification of Phenotype and Gene Expression in Human Retinal Pigment Epithelial ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2004;45(8):2786-2794. doi: 10.1167/iovs.03-0943.
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purpose. To use porcine lens capsule (PLC) as basement membrane for ARPE-19 cells and to characterize its effects on cell differentiation and gene expression.
methods. Postconfluent cultures of ARPE-19 cells were established on either porous polyester filters or PLC membranes and characterized by electron microscopy, immunocytochemistry, and transepithelial electrical resistance measurements. Metabolic activity was assessed by measuring phagocytosis of rod outer segments. mRNA populations of ARPE-19 cells grown on polyester and PLC membranes were compared by suppressive subtractive hybridization. Differentially regulated messages were subsequently identified by DNA sequencing and their altered expression confirmed by Northern or virtual Northern blot analysis.
results. Culture of ARPE-19 cells on PLC membrane induced the formation of apical microvilli and the ability to phagocytose rod outer segments. These culture conditions also led to enhanced junctional distribution of ZO-1 and occludin, the formation of polarized membrane domains, and a significant increase in transepithelial resistance. Gene expression was significantly altered by growth on PLC membranes and 29 differentially expressed transcripts were identified.
conclusions. Culture of ARPE-19 cells on PLC membranes resulted in a more differentiated phenotype and in expression of a specific set of transcripts encoding protein products that may affect epithelial differentiation, polarity and survival.
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