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)
Objective To investigate the effect of HGF on proliferation and migration in cultured human RPE cells. Methods Human RPE cells cultured in serum-free medium were treated with HGF(1，2，10，50，100 mu;g/L), and MT T assay was used to detect the growth of the cells; an in vitro wound healing model was used to count the number of cells that had entered the denudate area in RPE mig ration treated with HGF (1，2，10，50，100 mu;g/L) after 20 h. Results HGF(10,50,100 mu;g/L) increased proliferation rates of cultur ed human RPE (18.2 % to 34.8 %), and at a concentration of 50 mu;g/L on day 3 HGF induced the maximal increase of proliferation (Plt;0.01）; HGF showed effects on migration of 9.3 %, 113.0 %, 91.7 % and 50.3 % at the concentrations of 2,10,50, 100 mu;g/L, respectively. HGF stimulated t he best migratory response in RPE cells at a concentration of 10 mu;g/L (113 %). Conclusion HGF can induce the proliferation and obvious migration of RPE cells, consequently HGF was a mitogen and potent migratory factor for human cultured RPE cells. (Chin J Ocul Fundus Dis, 2001,17:307-310)
Objective To investigate the effects of applied electric fields on the migration of human retinal pigment epithelial (hRPE) cells, and probe the physiological effects and clinic significance of electric fields on the RPE cells during the wound healing process. Methods hRPE cells were clutured with Dubbeccolsquo;s modified Eagle medium (DMEM) (DMEM group ) or DMEM+10% fetal bovine serum (FBS) (DMEM+10% FBS group), and the cells unexposed to the electric fields were used as the control. After hRPE cells were exposed to direct current electric fields with the tension of 0,4, 6 and 8 V/cm, serial images were taken at 15 minuets intervals within 2 hours by photomicroscope to observe the migration of the cells, and then the migrating distances and angle between the cellular translocation direction and the field vector were measured. The results were analyzed by an image analyzer. Resutls The response of the hRPE cells cultured in DMEM group to the electric fields was not as obvious as that in DMEM+10% FBS group. The cells showed a tendency of cath odal migration when the field tension was added to 6 V/cm (the average cosine Phi; was compared with the normal control, P＜0.05.The effect of electric fields on cells clutured in DMEM+10%FBS group was ber than those in DMEM group. The long axes of the cells were perpendicular to the field line with the tension of 4 V/cm, and the cells continued migrating to the cathode, which was more obvious when the field tension increased (the average cosine Phi; was comparedwith the normal control, P＜0.05). After the polarity of the electric field reversed , the cells migrated to the opposite direction in accord with the direction of the cathode. Conclusion Applied electric fields can induce the cathode-directed migration of hRPE cells, which is positively correlated with the field tension and can be influenced by the serum. Physiological electric fields may be one of the inducing factors of cell migration at the early phase of healing process of wounded retina. (Chin J Ocul Fundus Dis, 2006, 22: 404-407)