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C. Zhu, J. Kurtz, N.C. Joyce; 2–D Proteomic Analysis of Cultured Human Corneal Endothelial Cells (HCEC) From Old and Young Donors . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1209.
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Purpose: To illustrate the similarity and difference in protein expression of cultured human corneal endothelial cells (HCEC) from different aged donors. Methods: Five pairs of donor human corneas with ages younger than 30–years–old (<30 yo), as well as another 5 pairs from donors older than 50–years–old (>50 yo) were obtained from National Disease Research Interchange and formed 2 age comparison groups. Primary culture and subcultures were performed following published protocols. Confluent passage 2 cells were rinsed with PBS to remove residual culture medium. Cell scrapers were used to remove cells from the culture plates. Harvested cells were centrifuged at 5K rpm for 10 min to form firm pellets. Bio–Rad Extraction Buffer III (ER3) was added and cells were homogenized for 1–2 min. Soluble proteins were harvested after centrifugation at 36K rpm at room temperature for 1 hr, then stored at –80°C until further analysis. Five samples from each age group were pooled and protein concentration determined by a modified Bio–Rad protein assay. 125ug of protein were loaded onto 17cm IPG strips with different pH ranges for iso–electric focusing (IEF). Proteins were then separated on19 cm Pre–Cast gels (8–16 % & 10–20% acrylamide) run at 1440 Vhr. After fixing and staining with Sypro–Ruby, protein spots were imaged by ProXPRESS, then analyzed using Nonlinear Dynamics PRO Finder 2005 version software. Results: Comparison of combined samples from the < 30 yo and > 50 yo groups showed similar protein patterns, as well differences. Analysis of protein separated on pH 3–10 and pH 5–8 IEF strips showed differences in approximately 10% of the protein spots between the two age groups. Conclusions: The majority of proteins were similarly expressed in confluent passage 2 HCEC from both young and older donors. However, age–related differences were also detected in relative protein levels, as well as in specific protein expression. These findings could lead to a greater understanding of important functional differences between HCEC from old and young individuals.
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