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H. Karring, I.B. Thogersen, G.K. Klintworth, J.J. Enghild, T. Moller-Pedersen; Characterization of Keratocyte Phenotypes using Proteomics . Invest. Ophthalmol. Vis. Sci. 2003;44(13):867.
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Purpose: The corneal keratocytes have a dynamic potential for proliferation and cellular transformation, and undergo phenotypic changes during corneal growth, repair, and disease. Increased knowledge on the various keratocyte phenotypes, their interactions, and transformation pathways is important for understanding corneal physiology and pathophysiology. Currently, there is no clear terminology on keratocyte phenotypes, and specific criteria have not been defined. Using proteomics, the purpose of the present study is to characterize 1) the keratocyte present within the normal human cornea, and 2) the derivative obtained from an explant culture. Methods: "Freshly-isolated-human-corneal-keratocytes" were obtained from six normal donors (aged 30-85 years) by stromal dissection and collagenase digestion for 4 hours. These cells were not exposed to serum. "Serum-cultured-human-corneal-keratocyte-derivatives" were obtained by explant culture of stromal samples and growth to third passage in Dulbecco's DMEM/F12 media containing 10% FBS. All cells were washed twice in PBS and lysed by sonication, and 2D gel electrophoresis was performed on the soluble fraction. After silver staining, all gel spots were analyzed using MALDI mass spectrometry. Results: The 2D gel analyses revealed more than 100 different proteins and isoforms in both the freshly-isolated and the serum-cultured cells. Distinct differences in the protein expression profile of the two phenotypes were observed. Furthermore, proteins not previously recognized in these cells were identified. Visit www.corneaproteomics.org for more information. Conclusions: Systematic identification and characterization of the protein expression profile by proteomics provide a rational basis for defining specific keratocyte phenotypes and their nomenclature.
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