Differential Protein Expression of Cultured Human Corneal Endothelial Cells From Old and Young Donors – A 2–D Proteomic Approach

Abstract

Purpose: : To compare the relative protein expression of human corneal endothelial cells (HCEC) cultured from young and older donors.

Methods: : Five pairs of human corneas from donors 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 3 cells were rinsed with HEPES isotonic buffer prior to scraping from culture plates. After removing the washing buffer via centrifugation at 5K rpm for 10 min, Bio–Rad Sequential Extraction Buffer III (ER3) was added to lyse the cells. Soluble proteins were harvested after centrifugation at 36K rpm at room temperature for 1 hr, and 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. 200ug of protein from each pooled sample was loaded onto 4–7 pH range 17cm IPG strips. Active re–hydration was performed for at least 12 hours prior to iso–electric focusing (IEF) to achieve optimal 1^st dimensional separation. 2^nd dimensional protein separation used 19 cm Pre–Cast gels (8–16 % acrylamide). Protein spots were fixed and stained with Sypro–Ruby, then imaged by ProXpress (Perkin Elmer). Nonlinear Dynamics Pro Finder 2005 version software analyzed the gel images. A pick–list was generated containing protein spots that showed a 2–fold normalized volume difference between the 2 samples. Matrix Assisted Laser Desorption Ionization Time–of–Flight Mass Spectrometry (MALDI–TOF MS) identified the proteins.

Results: : 457 spots from the young donor gel and 431 spots from the old donor group were detected and analyzed. From the young donor gel, 68 spots showed a 2 –fold increased normalized volume. Another 67 spots showed 2–fold decrease when compared with the gel from older donors. MALDI–TOF MS identified 24 proteins showing expression differences. These included specific cytoskeletal proteins and proteins supporting physiological activity, cell cycle regulation, or involved in protection against oxidative injury.

Conclusions: : Although the majority of proteins were similarly expressed in confluent passage–3 HCEC from both young and older donors, age–related differences were detected. Further identification of these proteins will greatly help us understand functional differences between HCEC from old and young individuals.

Keywords: cornea: endothelium • proteomics • aging