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N.V. Strunnikova, J. Flippin, P. Liu, E. Hoffman, K. Csaky; Use of Primary Retinal Pigment Epithelia (RPE) Cultures Derived From Healthy and AMD Affected Post–Mortem Donor Eyes for the Evaluation of Injury–Induced Differences in Gene Expression Patterns . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1153.
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Purpose: Combination of both environmental and genetic factors may be involved in the pathogenesis of AMD. Direct evaluation of differences between RPE derived from AMD or age–matched non–AMD was performed using primary human RPE (hRPE) cultures derived from post mortem donors to determine potential differences in genetic responses to a non–lethal in–vitro. Methods: hRPE cell cultures were derived from 55 years and older post mortem donors. Electron microscopy (EM) was used to assess ultra structural presence of AMD, graded in a masked fashion, from a punch biopsy obtained from the macula of each eye. The percent expression of cytokeratine 8/18, CD14 and CRALBP markers, as determined by FACS, western blotting and RT–PCT, was used to access cellular composition of the cultures. Selected passage 7–12 hRPE cultures were injured with nonlethal doses of oxidative stimulus hydroquinone (HQ) as determined by XTT evaluation for each culture. Gene expression patterns were examined using Human Genome 133 plus 2 GeneChips (Affymetrix) and verified by real–time PCR analysis. Results: Fourteen primary hRPE cultures were chosen for analysis. These included 7 cell lines in which the diagnosis of AMD was determined by the presence of Basal Laminar and Linear Deposits (BLD). In addition, only those cultures were chosen in which the percentage of cytokeratine 8/18 positive cells was between 60 – 95%. Microarray analysis showed that expression of several functional gene groups was affected in injured hRPE cultures from AMD donors, including matrix and adhesion proteins, injury related proteins, transcriptional regulators, and others. Analysis done by Expression Analysis Systematic Explorer (EASE) demonstrated that number of biological categories were overrepresented in the list of significantly changing genes following the injury. Conclusions: Primary hRPE cultures from AMD affected and age–matching donors could be used to determine differences in expression of genes involved in injury–induced wound repair, transcription, cell injury, matrix turnover, and stress response. However, these differences in gene expression were elucidated only following the nonlethal oxidative treatment highlighting the importance of environmental factors in pathogenesis of AMD.
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