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Lei Zhou, Jing Li, Siew Kwan Koh, M Setiawan, Hui Leng Khiu, Roger W. Beuerman; Quantitative Proteomic Analysis of Cellular Response to Hyperosmolarity in Human Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3809.
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© ARVO (1962-2015); The Authors (2016-present)
Tear hyperosmolarity is associated with dry eye disease. The aim of this study was to describe the effect of osmotic stress on human conjunctival epithelial cells using quantitative proteomic methods.
IOBA-NHC cells were cultured in DMEM:F12 medium (277 mOsm/kg), and subsequently challenged with hyperosmotic medium (377, 477 and 577 mOsm/kg) for 24 hrs. Cell viability was measured. iTRAQ (Isobaric tags for relative and absolute quantification) combined with two dimensional nano-liquid chromatography-tandem mass spectrometry (2D nanoLC-MS/MS) was used to profile the changes of protein expression in IOBA-NHC cells after hyperosmolarity shock. Western Blots were used to verify the proteomic results.
Among more than 600 identified proteins (95% confidence, Protscore ≥ 1.3) in iTRAQ experiments, we found a total number of 182 proteins that were differentially expressed in IOBA-NHC cells under osmotic stress (compared 577 mOsm/kg with control 277 mOsm/kg and using cutoff values: ratio > 1.35 or < 0.75). Eighty-eight proteins were overexpressed, whereas 93 proteins were down-regulated. Some differentially expressed proteins included inflammatory proteins, S100 A6 (1.60-fold increase) and alpha enolase-1 (1.42-fold increase), which have been previously identified as potential biomarkers for dry eye disease. Pathway analysis revealed that differentially expressed proteins were mainly involved in response to stress, apoptosis, cell cycle, cytoskeleton remodeling and DNA damage repair.
A quantitative proteomics approach provides a detailed picture of changes of protein expression of human conjunctival cells under osmotic stress. This information may lead to better understanding of the effects of hyperosmolarity on ocular surface in dry eye disease.
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