Study Of Porcine Retinal Pigment Epithelium Cell Replicative Senescence Stimulated By Blue Light

OBJECTIVE:Age-related macular degeneration (AMD) is the leading cause of severe vision loss and irreversible blindness over the age of 50y. Study of cytology and pathology indicated that retinal pigment epithelium (RPE) cell above the drusen could exhibit cell replicative senescence changes. The aim of these experiments is to observe the effects of blue light on porcine RPE cells replicative senescence and discuss the role of relationship between blue light and RPE replicative senescence in pathogenesis of AMD.METHODS:1. Primary porcine RPE culture was harvested with trypsin digestion. Cell morphology and characterization were assessed by phase contrast microscopy. The cells were identified with immunohistochemistry and immunofluorescence.2. The cells were seeded to 50ml flask after confluence.Since passage 3, the porcine RPE cells were placed under exposure of blue light source of LED which was designed and made by us. The blue light wave ranged was from 450nm to 500nm.The exposure time was one hour per day and the exposure intensity were (500±100) lux, (10004±200) lux, (20004±500) luxrespectively. The temperature of the surface of cells was between 36.5～7.5℃during exposure. The control group was encapsulated by black paper during exposure.3. The cells were seeded at a density of 2×10⁵ cells/ml into 24 well culture plate for 24h and then fixed by 4% paraformaldehyde to detect beta-galactosidase activity from passage 3 to passage 6.RPE cells replicative senescence was defined of blue particles in plasma of cells. Percentage of positive cells were counted in 5 selected random high power microscope fields and repeated thrice.4. The cells were seeded at a density of 1×10⁵ cells/ml into 96 well culture plate for 24h and followed by MTT assay to detect cell proliferative activity. Each group detected in 6 wells, and repeat thrice.5. All data were showed with mean±standard deviation((?)±s) and were dealt by SPSS 12.O. After test of normality and homogeneity test for variance, multiple factor analysis of variance were used if equal variance assumed followed by multiple comparisons with test of LSD-t. The mean difference was significant at the level of 0.05.RESULTS:1. This primary culture resulted in cells with well-preserved morphology, some cells grew in colony. The cells morphology was irregular, but they could be identified shape of sexangle when reaching confluence. The cells were vigorous for further passage and resuscitation. The result of immunohistochemistry and immunofluorescence were positive of the antigen of anti-keratin.2. Different intensity of blue light exposed on porcine RPE cells from passage 3 to passage 6 .The number of the positive cells with blue particles increased as the function of exposure intensity while the increased the activity of SA-13-Gal. The value of F is 144.81,Ρ＜0.01 within the different light exposure intensity. Except for the difference between high light intensity and middle light intensity, every other two groups have statistics significance (Ρ＜0.05). Between different passage, the value of F is 23.41,Ρ＜0.01 within the different passage. Except for the difference between passage 4 and passage 5 , different between every other two passage have statistics significance(Ρ＜0.05).3. Under the same exposure intensity the cell proliferation activity decreased while the passage increased (F＝31.16,Ρ＜0.01), Except for the difference between ??middle intensity and low intensity ,every other two groups have statistics significance(Ρ＜0.05). The cell proliferation activity decreased with the increased light intensity(F=86.52,Ρ＜0.01), every two passage have statistic significance (Ρ＜0.05)CONCLUSION:These experiments establish the culture of primary porcine RPE cells, and the cells maintain active proliferation,and positive to anti-keratin antigen by immunohistochemistry and immunofluorescence. These experiments get the similar results using the same methods with human RPE ceils culture, but porcine RPE cells are easier to obtained and convenience to operate, and can be used to do some research work. This research observed the effects of blue light on porcine RPE cells replicative senescence with blue light device made by us. The replicative senescence of porcine RPE cells cultured in vitro are positive correlation with blue light intensity and passage times. The relative research may be helpful for understanding of the AMD relative cytobiology pathogenesis. These experiments firstly combine blue light, RPE cell replicative senescence and pathogenesis of AMD together and provided held evidence for blue light induced RPE cells replicative senescence in pathogenesis of AMD. Further understanding of these relationships could facilitate methods for prevention and cure AMD in clinical trial. Theses experiments apply replicative senescence into ophthalmology's clinical and experimental study which will benefit to develop a new experimental field of ophthalmology.