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)-alpha; for 24 hours, 1 and 6 mu;g/ml lipopolysaccharide (LPS) for 11 hours, 7 ng/ml TNF-alpha; 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 b 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-alpha; or LPS increased, the fluorescence intensity decreased（Plt;0.01）, and after exposed to 30% supernatant of THP-1 cells, weaker fluorescence intensity was detected (Plt;0.001). Pretreatment with 50 mu;mol/L PD098059 for 2 hours partly inhibited the effect of THP-1 cells supernatant. After exposed to 30% supernatant of THP-1 cells for 3 hours, the number of attached cells decreased compared with the controls（Plt;0.05）. Conclusions TNF-alpha; and LPS down-regulate the expression of syndecan-1 in cultured human RPE cells. The supernatant of THP-1 cells down-regulates the expression of syndecan-1 and lessens the cells attaching, which is at least mediated by ERK 1/2 pathway. (Chin J Ocul Fundus Dis, 2006, 22: 113-116)
ObjectiveTo observe the effect of triamcinolone acetonide(TA) on activation and barrier function of human retinal pigment epithelium （RPE）.MethodsARPE-19 cells were cultured in 96well tissue culture plate. Four weeks later, TA with different concentration (0.02 and 0.05 mg/ml)was added to the cells and culture for 3 or 7 days. The activation of ARPE-19 cells was assessed by methyl thiazolyl tetrazolium (MTT). ARPE-19 cells were cultured on polyester microporous filters for 4 weeks, and the transepithelial resistance (TER) was recorded. TA (0.02 and 0.05 mg/ml) was added to the culture fluid respectively, and after cultured for 1 week TER was measured again. The RPE permeability was detected by enzymelinked immunosorbent assay (ELISA) with horse radish peroxidase as the tracer. ResultsIn the culture fluid with 002 mg/ml TA cultured for 3 or 7 days, the average survival rate of ARPE-19 cells was 93.70% and 90.63% respectively, without statistic difference compared with the control (P=0.147, 0.091). While in the 0.05 mg/ml TA group after cultured for the same duration, the activation of ARPE-19 cells decreased significantly compared with the control (with the average survival rate of 87.75% and 88.98%; P=0.025, 0.043). One week after cultured with TA, TER decreased significantly while permeability improved obviously in the 2 TA groups compared to the control (Plt;0.001; 0.001lt;Plt;0.05).ConclusionTA may decrease the activation of and destroy the barrier function of ARPE-19 cells. (Chin J Ocul Fundus Dis, 2005,21:237-239)
Objective To investigate the effect of hypericin on the activity of protein kinase C (PKC) in cultured human retinal pigment epithelium (RPE) cells in vitro.Methods RPE cells were cultured in standard medium with 10% serum concentrations containing 0.5 to 5.0 μmol/L hypericin with or without preincubation of phorbol 12-myristate 13-acetate (PMA). The activities of cytosolic PKC (c-PKC) and membranous PKC (m-PKC) were assayed by PKC kit. Results The original activities of c-PKC and m-PKC of RPE cells were (35.34±4.10) pmol·min-1·mg-1and (62.52±8.80) pmol·min-1·mg-1.The activity of c-PKC in RPE cells with PMA preincubation decreased rapidly in 5 minutes, with a subsequent slow decrease after 20 minutes and a decrease to 18% of the activity of c-PKC in RPE cells without PMA preinubation after 60 minutes. While the activity of m-PKC in RPE cells with PMA preincubation increased gradually after 5 minutes and reduced after reached the peak at 40 minutes, and then returned to baseline after 60 minutes, eventually decreased below 30% of the control group. When RPE cells were cultured with PMA for 48 hours, the activities of c-PKC and m-PKC were hardly detectable, while RPE cells were cultured with both PMA and hypericin, hypericin could counteract most of down-regulation by PMA. Conclusion Hypericin may inhibit the translocation of PKC in RPE cells,change the activity of PKC, promote the apoptosis of RPE cells likely,and then prevent proliferative vitreoretinopathy. (Chin J Ocul Fundus Dis，2003，19：55-58)
Objective To investigate the effects of QUE on proliferation and DNA synthesis of cultured retinal pigment epithelium(RPE) cells with or without EGF. Methods With or without EGF, cultured RPE cells were treated with QUE by various concentrations(200,100,50,1mu;mol/L) and with QUE 200mu;mol/L at different times(24-168 hr), cells proliferation and DNA synthesis were evaluated by cell count method and the uptake of thymidine. The viability of cells was determined by trypanblue exclusion. Results The best concentration of QUE which inhibits proliferation and DNA synthesis of PRE cells was 200mu;mol/L. The significant inhibition effect of QUE occurred at 48hr, and the best inhibition of QUE occurred at 96hr. QUE had more powerful effect of antiproliferation on RPE cells, and the viability of RPE cells was over85%. Conclusion The results suggested that QUE could inhibit the proliferation of RPE cells in a dose-dependent and time-dependent manner, especially inhibit the proliferation induced by EGF stimulating. QUE had no cyto-toxic effect on RPE cells cultured in vitro. (Chin J Ocul Fundus Dis,1999,15:27-29)
OBJECTIVE:To observe the effect of dexamethasone to intracellular free Ca2+ of frozen RPE cells. METHODS:The cultured human RPE cells were frozen for 30s at --70deg;C. The RPE cells were loaded with Fura-2/AM and analyzed using a digital imaging microscopy system,the effect of dexamethasone to intracellular free Ca2+ was measured at a serial concentration of 40, 60,100,150,200mu;g/ml. RESULTS:The concentration of intracellular free Ca in frozen human RPE cells was increased to 18.6%~29.8% by dexamethasone at concenlration of 40mu;g/ml~60mu;g/ml,while was decreased to 28.4%~35.2% at 150mu;g/ml~200mu;g/ml. CONCLUSIONS:Effect of dexamethasone showed two aspects of effect to frozen cultured human RPE ceils,that it was inhibitor at high concentration and stimulator at low concentration （Chin J Ocul Fundus Dis,1997,13: 86-88）