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Sung Rhan Weon, Jaime Chew, Queenie Tan, Veluchamy A. Barathi, Roger W. Beuerman; Regulation of Pigment Epithelial Derived Factor (PEDF) and Thrombospondin 1(THBS 1) in Mouse Retinal Pigment Epithelial cells under Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4439.
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To investigate the effects of oxidative stress on expression of pigment epithelial derived factor (PEDF) and thrombospondin 1(THBS 1) on cultured mouse RPE cells.
Primary RPE cells were harvested by trypsinzation from Balb/c mouse posterior segments. Cultured mouse RPE used were between passages 2-4. The RPE cells were plated into 60mm culture dishes at concentration of 6x 105 /2ml and incubated under normoxic conditions (95% air/5% carbon dioxide), allowing them to attach over night. The next day, samples were transferred to a hypoxic incubator to induce oxidative stress with hypoxic conditions (95% nitrogen, 5% carbon dioxide) for 24 hrs, they were then exposed to normoxic conditions. Control samples remained under normoxic conditions. After harvesting the cells at various time intervals (0,24,36,48,60 and 72 hr), the amount of RNA and protein were analyzed by RT-PCR, immunobloting and ELISA assay respectively.
RT-PCR results showed that the effects of oxidative stress was more prominent in transcript levels of PEDF than THBS1. After 24 hr of exposure to hypoxic conditions, there was 4-fold down regulation of PEDF compared to 2-fold down regulation of THBS1. THBS 1 recovered quickly after normoxia was applied and rebounded to more than 2-fold up-regulation at 72 hr. In contrast PEDF recovered more slowly and reacheda maximum after 60-72 hr. Immunobloting and ELISA assay showed similar expression patterns for PEDF and THBS.
Although PEDF and THBS 1 both have a role to protect against neoavascularisation, the significant up-regulation of THBS1 with restoration of normoxic conditions may have an important mechanistic effect to protect from oxidative stress.
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