Effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial cells / 中华眼底病杂志

ABSTRACT

Objective To investigate the effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial (RPE) cells and the signal transduction pathway. Methods Reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of syndecan-1 mRNA and protein in normal RPE cells. The expression of syndecan-1 in RPE cells stimulated by different cytokines was detected and quantitatively analyzed by image process of immunofluorescence. The stimulation included 7 and 35 ng/ml tumor necrosis factor (TNF)-? for 24 hours, 1 and 6 ?g/ml lipopolysaccharide (LPS) for 11 hours, 7 ng/ml TNF-? for 0 to 24 hours (once per 2 hours, and 13 times in total), and 30% supernatant of monocyte/macrophage strain (THP-1 cells) for 3, 14 and 43 hours. The effect of 30% supernatant of THP-1 cells was assayed after pretreated by PD098059 [the specific inhibitor of extracellular signal regulated kinase(ERK) 1/2] for 2 hours. After exposed to 30% supernatant of THP-1 cells for 3 hours and treated by 0.25% trypsin for 5 minutes, RPE cells attaching was evaluated by methyl thiazolyl tetrazolium assay. Results In normal human RPE cells, expressions of syndecan-1 mRNA and protein were detected, and strong syndecan-1 positive yellowish green fluorescence was found in the cell membrane and cytoplasm while light green fluorescence was in the nucleus. As the concentration and stimulated time of TNF-? or LPS increased, the fluorescence intensity decreased(P