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J.J. Peairs, P. Yang, Z.J. Zavodni, J.N. Ebright, C. Bowes Rickman, G.J. Jaffe; Human RPE Cell Survival Factor Gene Expression in situ Compared to Expression in vitro . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5368.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Retinal pigment epithelial (RPE) cells play an important role in the potentially blinding diseases of age–related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). RPE cell cultures are useful for determining changes, caused by oxidative stress or growth factors, in the expression levels of survival factors that can govern whether a cell survives or dies. To ensure that basal survival factor mRNA expression in cultured human RPE cells reflects gene expression in their normal monolayer in situ, we examined survival factor mRNA expression in samples of freshly isolated RPE cells from human donor eyes. Methods:RPE cells from six human donor eyes were pooled using a novel isolation technique. An ‘RPE–enriched’ sample was prepared by first removing the peripheral retina with a 4 mm trephine punch and collecting the underlying RPE/choroid tissue. Then, RPE cells were carefully brushed and washed from Bruch’s membrane and the retina, and the resulting RPE cell suspension was analyzed with light microscopy. The cells were spun down into an ‘RPE–enriched’ pellet, which then went through a melanin–removal step before being used for RNA isolation. Samples from ‘RPE–enriched’ and cultured RPE cells were analyzed using gene–specific primers with real–time RT–PCR. Results: For nearly all of the survival factors examined, basal in situ mRNA expression was observed at levels similar to those found in cultured human RPE cells. Bcl–xL, which showed the highest expression of any survival factor in cultured RPE cells, also showed the highest expression in situ, and at a quantity very similar to that found in cultured RPE cells. Similarly, c–IAP1, cFLIP, and TRAF–2 were found at intermediate levels (relative to the housekeeping gene, GAPDH) in both cultured RPE cells and in situ, TRAF–1 and cIAP2 were found at low levels in both samples, and Bcl–2 and A1 were found at very low levels in both samples. Survivin, the only survival factor that did not show parallel expression, was expressed at intermediate levels by cultured RPE cells but was not detected in situ; it has a constrained distribution in normal cells and is usually detected in neoplastic cells. Conclusions: There is good correlation between human in vitro and in situ survival factor expression. The human RPE culture model will be useful to study normal RPE survival factor expression, and in conditions that simulate ocular disease.
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