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A. Ando, Y. Mitsuma, T. Katano, S. Ito, T. Nishimura, K. Takahashi; Proteomics Analysis of Cultured Human Retinal Vascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1883.
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© ARVO (1962-2015); The Authors (2016-present)
Several different anti-glaucoma agents, especially prostaglandin analogues (PGs), have been reported to have a retina blood flow improvement effect, though the mechanism remains unclear. We used proteomics analysis with two-dimensional gel electrophoresis (2D-PAGE) to determine how PGs influence RVE cells.
Cultured human RVE cells (ACBRI 181, Applied Cell Biology Research Institute) were grown on attachment factor in CS-C culture medium. Following quantification with a 2-D Quant Kit, whole protein was extracted from the cells at sub-confluence with cell lysis buffer (including 8 M urea), then 100 µg of each sample was loaded onto ImmobilineTM Dry Strips (pH 3-10). Next, isoelectric electrophoresis (first dimension) was performed using gradient increased voltage to 8000 V with an IPGphor (General Electric). Following isoelectric electrophoresis, second dimension electrophoresis with ExcelGelTM 2-D (12.5%) was performed. Gels were stained with Coomassie Brilliant Blue and the numbers of separated protein spots were examined using PDQuest software (Bio-Rad). Furthermore, the gene expression of prostaglandin F2alpha (FP) receptor in RVE cells was investigated by RT-PCR.
RT-PCR revealed gene expression of the FP receptor. Protein extracted from the RVE cells was separated by 2D-PAGE into 205 separate spots.
Proteomics analysis using 2D-PAGE was helpful to examine changes in the protein profile of RVE cells for elucidation of the mechanism(s) of retinal blood flow improvement by anti-glaucoma agents.
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