| Purpose. Whether mitochondrial permeability transition (MPT) participates in blue light-induced damage to human retinal pigment epithelium (RPE) cells is still unclear. The purpose of this study is to investigate the signal transmission of blue light-induced injury and whether Gelsolin gene has the protective role.Methods. Human RPE cells were exposed to various intensity blue light (wave length 470-490nm) for various duration. Phototoxicity was quantified at various periods after expose by staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure. Transmission electronmicroscopy was used to determine the changs of human RPE cells. Mitochondrial membrane potential (△Ψm) was measured by rhodamine 123 staining and subsequent flow cytometry. Cytochrome C and caspase-3 activity were assayed by ELISA. Human Gelsolin full length cDNA was amplified from total RNA of human latissimus dorsi tissue using RT-PCR protocol. Gelsolin cDNA was clonedinto the eukaryotic expression vector pcDNA3.1(+) by 5' -HindIII and 3' -EcoRI sites, and analyzed with endonuclease and sequencing. Human RPE cells were transfected with the plasmid DNA encoding Gelsolin. The Gelsolin protein expression was detected through immunocytochemistry with a specific polyclonal antibody against the Gelsolin protein, or was confirmed by RT-PCR, By which the mRNA of Gelsolin in RPE cells transfected with the plasmid DNA encoding Gelsolin was detected.Results. RPE cells transfered of culture changed from multi-angle shape to oval-shape, anomalous shape or fusiform shape. Blue light caused activation of apoptosis in human RPE cells, indicated by characteristic morphologic. TUNEL-positive labeling included cell shrinkage, membrane blebbing, apoptotic body, condensation and fragmentation. Mitochondrial swelling, the extinction of inner mitochondrial membrane ridge, the dilation of the rough endoplasmic reticulum and the increase of the lysosome were also observed in transmission electronmicroscopy. A significant increase of apoptotic and necrotic percentages was consistent with higher light intensity; Apoptotic percentage increase was the main response to shorter exposure of blue light, whereas necrotic percentage increase to longer exposure; The apoptotic response was more pronounced during 6 and 12 hours prolongation of post-exposure culture, but more apoptotic necrosis or necrosis were found after post-exposure 24 hours.The change of △Ψm was confirmed by using rh123. When human RPE cells were exposed to blue light, the more pronounced decrease of △Ψm was consistent with the light intensity and exposure duration; By 6 hours prolongation of post-exposure culture, the decrease of △Ψm was observed, lasting 48 hours. The concentration ofcytochrome C was detected, when human RPE cells exposed blue light 6 or 12 hours respectively, No significant changes were found at 6 and 12 hours prolongation of post-exposure culture. But a significant increase was found treated with post-exposure 24 and36 hours in two groups. The latter was more significant increase than the former at post-exposure 24 hours. The activity change of caspase-3 was not found in two groups. Endonuclease analysis revealed that the insert segment in the resultant recombinant pcDNA 3.1 (+) was Gelsolin cDNA.Conclusions. Blue light is able to cause human RPE cells damages in vitro, the patterns include apoptosis, apoptotic necrosis and necrosis. The extent is dependent on exposure intensity and duration. Blue light exposure over threshold can induce apoptosis in human RPE cells, probably by triggering the mitochondrial permeability transition, which results in swelling of the mitochondria, decrease of △Ψm and release of cytochrome C. But the change of Caspase-3 activity is not detectd. In the study, we successfully constructed Gelsolin expression vector pcDNA3.1-Gelsolin. |
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