Purchase this article with an account.
L. Geng, L.V. Del Priore, H.J. Kaplan, T.H. Tezel; Change in Human Retinal Pigment Epithelium (RPE) Proteome Cultured on Different Matrices . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1385.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate alterations in human RPE proteome cultured on bovine corneal endothelial matrix (ECM) and tissue culture plastic (TP).
Three different lines of adult human RPE were established on bare TP and ECM–coated dishes (donor ages: 48 and 59, 70). RPE cells were serially cultured up to the 7th passage. 30 µg of RPE protein was extracted from the primary, first, third and seventh passage cells and focused using pH 3–10 7.7cm IEF strips. Proteins were further separated in the second dimension on 4–12% Bis–Tris gels. After Sypro Ruby staining spots of interest were cut and peptide analysis was done using mass spectrometry. Quantification of protein expressions at different passages on ECM and TP was done using Phoretix Expression 2D software and compared with the primary RPE proteome.
Significant changes occurred in the primary RPE proteome by culturing. However, quantitatively proteome changes on TP and ECM were similar. Only 27.7% of the primary proteome on TP and 31.4% on ECM were preserved in the first passage, p=0.54). As the passage number increase the number of proteins disappearing from the primary RPE proteome increased (6.9% vs.11.9%, 17.0% vs 17.6%, 12.6% vs. 20.1% for the 1st, 3rd, and 7th passage on TP and ECM, respectively). Regardless of the matrix the production of hemoglobin, adenylate cyclase, cxorf5 and immunoglobulin chains decreased by culturing. Additionally, on TP there was significantly less palladin, TNF–induced transmembrane protein, and lamin B2. On ECM, synaptosomal–associated protein, neurofilament and elastase 3 were expressed in significantly less quantities.
RPE cell proteome changes significantly by culturing. Although quantitatively no change was detected in cell protein expression on TP and ECM, qualitative changes indicate that the matrix exerts a significant effect on RPE proteome.
This PDF is available to Subscribers Only