Protective Effects Of Erythropoietin On Human Retinal Pigment Epithelial Cells

Objective To explore the molecular mechanism of human retinal pigment epithelium cells injury and the protective effects of erythropoietin on human retinal pigment epithelium cells injury. This primary results might provide a theoretical basis of erythropoietin's clinical application for retinal diseases. Methods 1. The human fetal retinal pigment epithelium cells were isolated and primary cultured to passage 4 or 6.Immunohistochemical method was applied to identify retinal pigment epithelium cells. The human retinal pigment epithelium cell line, ARPE-19 were passage cultured. The expression of erythropoietin and erythropoietin receptor were detected. 2. The retinal pigment epithelium cells oxidative injury models were established. MTT cell viability assay was applied to detect cells viability. The expression of erythropoietin and erythropoietin receptor were detected by immunohistochemical method and Western blot method. 3. Apoptosis was assessed by annexin V-fluorescein isothiocyanate -Propidium iodium(annexin V-FITC-PI )staining method and DNA ladder method. Reverse transcription- polymerase chain reaction(RT-PCR) ,Western blot and immunohistochemical methods were applied to detect the expressions of clusterin, Caspases-1, Caspases-3, Caspases-9. 4. To add extrinsic source erythropoietin to the cell culture solution as the erythropoietin intervention group. Annexin V -PI staining, RT-PCR and Western blot methods were applied to detect the changes of cells oxidative injury, apoptosis and expression of apoptosis-related-factors after erythropoietin intervention, to observe the protective effects of erythropoietin on cultured retinal pigment epithelium cells in vitro. Results 1. The primary cultured fetal human retinal pigment epithelium cells presented sexangle cells, contenting melanin granules in cytoplasm. The melanin granules decreased along with cell passage. When the cells passaged to 3 or 6, the cultured cells presented short fusiform shape or fusiform shape, few melanin granules or none in cytoplasm. Identified retinal pigment epithelium cells by immunohistochemical method showing that the expression of cytokeratin was positive and the expression of glial fibrillary acidic protein was negtive. The adult retinal pigment epithelium cells (ARPE-19 cell line) presented long fusiform or triangle shape, monolayer adherence growing. The nucleus was lucency, large and round, having 1 or 2 chromatospherite.The endochylema was abundant. The cell boundary was clear. Detected by immunohistochemical method, Erythropoietin and erythropoietin receptor expressed on normal human fetal and adult retinal pigmentepithelium cells. 2. In oxidative injury model group, cells viability decreased obviously. Erythropoietin and erythropoietin receptor expression in oxidative stress group increased. 3. The oxidative injury might caused retinal pigment epithelium cells death. Detected by Annexin V-PI staining method, injured by 600umol/L hydrogen peroxide, the way of cell death mainly was cell apoptosis. Detected by RT-PCR and Western blot methods, the expression of clusterin changed along with the action time of hydrogen peroxide. Detected by immunohistochemical methods, the expression of Caspases-1, Caspases-3 and Caspases-9 were increased along with the prolongation action time of hydrogen peroxide. 4. Compared with the oxidative injury group, in the erythropoietin intervention group, retinal pigment epithelium cells viability increased, the apoptotic cells percentage decreased, the expression of clusterin increased and the expression of Caspases-1, Caspases-3 and Caspases-9 decreased. Conclusion Erythropoietin and erythropoietin receptor expressed on normal human fetal and adult retinal pigment epithelium cells, might increased induced by hypoxia and oxidative stress. The way of death of retinal pigment epithelium cells in cumulatly oxidative injury mainly was apoptosis. The mechanisms of apoptosis might include decreased cells viability, decreased expression of clusterin and provocation of Caspases apoptotic pathway. Erythropoietin might protect retinal pigment epithelium cells by increasing cells viability and decreasing cell apoptosis.