Effect Of Vitamin E On Replicative Senescence Stimulated By Blue Light In Human Retinal Pigment Epithelium Cell

Purpose:Replicative senescence is now understood to be a phenomena exhibited by most nontransformed cells in vitro, which may relate to some age related diseases. This study is to observe the senescence related beta-galactosidae (SA-β-Gal) of cultured senescent human retinal pigment epithelium(RPE) cells in vitro stimulated by different intensity of blue light with fixed wave length and the effect of Vitamin E(VitE) to this. To provide the relative results for the relation of replicative senescence with age related diseases just like age-related macular degeneration (AMD) .Methods:1. Human RPE cell culture in vitroFresh human eyeball from accidental death for experimental material, trypsin digestion method for human RPE cell primary culture. Observe under microscope, identified with immunohistochemistry.When cells fused, passage as 1:2～1:3, cultured with 10%FBS solution. Passage every 4～7 days after that, observe cell growth state and drow the grow curve.2. Replicative senescence stimulated by blue light and the effect off VitEFrom passage 2, add VitE in the culture solution, make different groups' clutrue solution final concentration of VitE at four levels: 100μmol/L,50μmol/L,10μmol/L and 0; meanwhile all these cells were divided into four group with different blue light intensity of cell surface, that were: (2000±500) lux, (1000±200) lux, (500±100) lux and 0. Blue light's wave length was 450～500nm. All the cells exposd to the light for 1 hour a day until the passage 5, observe cells' morph and growth condition during this. Make the cell seeded in culture plate, culture for24 hours, then fix for 30 minutes, and compose with 0.1%TritonX-100 for 20 minutes at ordinary temperature. Add SA-β-Gal detect solution at ordinary temperature, away from light for 24 hours. Observe if there were any blue paniculate in the cells; Make the cells seeded in culture plate, keep at ordinary temperature, away from light for24 hours. Add DMSO, shake for 10 minutes, detect OD value at 600nm. 3. Statistics methodWith SPSS13.0, after normality test and homogeneity test for variance, for two way ANOVA, then SNK test for multiple comparison. P<0.05 have statistical significance.Results:1. Morphologic character of human RPE cellPrimary cell adhere after 24～48 hours and get flat, stretch out parapodium 4～5 days later. The cells is irregularity polygon, the round transparent area in the center is the cell nucleus. Many cells have two or more nucleus, cell colony could be seen in the division phase. Immunohistochemistry cytokeratin detection show positive reaction. Cell fuse in 5～10 days. With the passage, growth speed get slow and melanin granule in kytoplasm get less, color of cell get close to transparence. Senescent cells was bigger and flater, most of them change from polygon shape, cell process emerge, morphologically similar to fibroblast. After the SA-β-Gal detection, positive cell was stained to blue.2 Replicative senescence stimulated by blue light and the inhibit effect of VitESA-β-Gal detect: with the light intensity increase and VitE concentration decrease the SA-β-Gal positive rate increase. Different group of the light's effect were all have statistical significance (P<0.01); different VitE concentration also have statistical significance (P<0.01) except the 10μmol/L and 0 (P>0.05).MTT detect: with the light intensity increase and VitE concentration decrease, cell reproductive activity decrease. The effect of (2000±500) lux, (1000±200) lux and (500±100) lux have no statistical significance (P>0.05); different VitE concentration have statistical significance (P<0.01) except 100μmol/L and 50μmol/L (P>0.05).Conclusions:1. With the method in this study we culture human RPE cells in vitro successfully, passage, freeze and anabiosis based on this, all were satisfacted.2. Blue light in a range of intensity could stimulate replicative senescence of human RPE cell, with the light intensity enhance, rate of SA-β-gal positive cells increase.3. VitE could inhibit the blue light effect to the cell, inhibit effect enhance with the VitE concentration.4. Proved that oxidative damage cause by blue light is one of the mechanism of replicative senescence of human RPE cells.