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S.M. Hauck, S. Suppmann, M. Ueffing; Proteomic Profiling of Primary Retinal Müller Glia Cells Reveals a Shift in Expression Patterns Upon Adaption to In Vitro Conditions . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4561.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cultured primary Retinal Müller Glia cells (RMG) have been widely used as a cell system to study their role in retinal biology especially with respect to their anticipated role in supporting photoreceptor survival and function. Identification of the underlying molecular mechanisms and factors presented or released by RMG may provide a rational basis for the treatment of blinding retinal degenerations. In order to determine those conditions of isolated primary RMG cells that correspond best with their role in retinal physiology, and thus establish a basis for a genome-based analysis of RMG specific gene expression, 2 DE based protein fingerprints were established allowing to monitor the dynamics of protein expression during prolonged culturing. Methods: Isolated primary porcine RMG were enriched and maintained in vitro for different periods. Purity of the RMG cultures was determined by immunohistochemistry with antibodies against Vimentin and Glutamin-Synthetase. Freshly isolated RMG and RMG after 3, 8 or 14 days in vitro were homogenized and their proteins separated at high resolution by two dimensional electrophoresis (2 DE). Isolated proteins were identified by matrix –assisted laser desorption ionisation time of flight (MALDI-TOF) fingerprint Results: Comparison of the 2 DE maps from feshly prepared RMG with the maps of RMG from different timepoints in culture reveal stability of RMG protein expression during initial culturing but profound differences in protein expression after day three in culture. Proteins involved in specific physiological functions of RMG such as glycolysis, transmitter-recycling, CO2-siphoning, phototransduction cycle and detoxification are downregulated or shut down whereas cytoskeleton proteins are upregulated during culture. Conclusions: At the protein expression level, cultured RMG show loss of RMG specific proteins over time. The profound changes of the proteome reflect transdifferentiation from a multifunctional, higly differentiated phenotype to a dedifferentiated monolayer of cells in culture.
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