Abstract
Purpose: :
To investigate biological characteristics of cultured porcine RPE cells in vitro after internalization of A2E and the effect of visible light with different wavelengths on cultured A2E-laden porcine RPE cells in vitro.
Methods: :
The cultured RPE cells were identified by immunohistochemical staining with anti- human keratin and RPE65. A2E were synthesized from lipofuscin fluorophore in vitro. A2E with different concentrations (50, 75,100µmol/L) were delivered to cultured porcine RPE cells in media for internalization. Cells were observed under fluorescence microscope at 30,45,60,90 minutes respectively after A2E phagocytosis and the viable cells were detected by MTT assay.Cultured porcine RPE cells that had accumulated A2E were divided into 7 groups according to visible light exposure with 405,440,475,535,575,630,680 nm wavelengths respectively and same intensity. Apoptotic cells were detected by annexin V-fluroresein isothiocyanate (FITC)/ propidium iodine (PI) flow cytometry.
Results: :
When A2E was delivered to RPE cells in culture, it accumulated intracellularly. Internalized A2E presented as autofluorescent granules showing a perinuclear distribution. MTT assay and grayscale scanning fluorescence results showed that the variable cells showed dose-response with different concentrations of A2E uptake by the cells and 50 µmol / L at 60 minutes after A2E phagocytosis was the best experimental condition. Apoptosis of cultured porcine RPE cells that had accumulated A2E was induced by visible light with short wavelength (under 530nm). Light wavelength around 475nm had the most damage effect at the same light intensity.
Conclusions: :
A2E-laden RPE cells with the concentration of 50µmol/L and the time of 60 minutes after A2E phagocytosis might be injured by light with specific wavelength. The different damage effect might be related to the light spectrum range.
Keywords: retinal pigment epithelium • phagocytosis and killing • protective mechanisms