Experimental Study Of The Lycium Barbarum Polysaccharide On The Role Of Retinal Pigment Epithelial Cells

Objective:Establish model of age-related macular degeneration though blue light burning human retinal pigment epithelium cells cultured in vitro,and then culture the cells by Dulbecco′s modified eagle′s medium with high glucose mixed with different densities of ycium barbarum polysaccharide to explore the effects of lycium barbarum polysaccharide on retinal pigment epithelium cells and explore the feasibility of AMD prevention and treatment of LBP.Methods:1. Observe the situation of human retinal pigment epithelial cell(hRPE cell) cultured in vitro thougth inverted phase contrast microscope. 2. Prepare medium with final concentrations of 10mg/ml, 1mg/ml, 0.1mg/ml, 0.01mg/ml LBP to clture hRPE cells in vitro, and use CCK-8 to choose the safe LBP concenrations. 3.The hRPE cells were divided into 5 groups: A group were normal cells-control group; B group were hRPE cells burned by blue light-light injury group; C group were hRPE cells cultured with medium mixed with final concentrations of 1mg/ml and induced damage by blue light; D group were hRPE cells cultured with medium mixed with final concentrations of 0.1mg/ml and induced damage by blue light; E group were hRPE cells cultured with medium mixed with final concentrations of 0.01mg/ml and induced damage by blue light. The hRPE cells of B-E groups were exposed to (2000士500)LUX intensity blue lihgt (wave length 470nm-520nm) for 12 hours, and the cellular morphology were observed by inverted phase contrast microscope. Use Annexin V-FITC/PI kit, mitochondrial membrane potential detection kit and mitochondrial reactive oxygen species assay kit to detecte by flow cytometry of mitochondrial reactive oxygen species, mitochondrial membrane potential and apoptosis, and futher study of the effect of LBP on hRPE cells exposed to blue light-induced damage and the possible mechanism.Results:1.Thougth inverted phase contrast microscope: hRPE cells cultured in vitro were spindle or polygonal, adherent monolayer growth, and cell borders were clearly. 2. Thougth test of CCK-8: (1) There were statistically significant differences about the OD values between cells with medium mixed with final concentration of 10 mg/ml LBP and nomal retinal pigment epithelial cell group(P<0.05);(2) The OD values of cells with medium mixed with final concentrations of 1 mg/ml LBP,0.1mg/ml and 0.01 mg/ml LBP were no significant differences with nomal retinal pigment epithelial cell group(P>0.05). 3.Observe cells of group A-E by inverted phase contrast microscope after establishing the blue light-induced damage model: Cells of group A were spindle or polygonal, adherent monolayer growth, and cell borders were clearly. Some of cells of group B were shrinkage, rounding, refraction changes, and around the cells coule be seen bright ring; nucleus shrink, split into multiple or a nuclear margination; there were cell debris suspension cells in the culture medium. 4. Annexin V-FITC/PI: (1) The least number of apoptotic cells in group A.(2)The most number of apoptotic cells in group B.(3)The number of apoptotic cells of groups C-E were specifically significant differences with group B(P<0.05);(4)The number of group C and D were no significant different with group A(P>0.05). 5. Detection of mitochondrial membrane potential: (1) Group A had the most number of positive cells;(2) Group B had the least number of positive cells;(3) The number of positive cells of groups C-E were specifically significant differences with group B(P<0.05); (4)The numbers of group C and D were no significant different with group A(P>0.05). 6.Detection of mitochondrial reactive oxygen species: (1)Group A had the maximum fluorescence level; (2) The fluorescence level of groups C-D were specifically significant differences with group B(P<0.05); (3)The fluorescence level of group E was no significant different with group B(P>0.05).Conclusions:LBP can inhibit apoptosis in blue light-induced damage hRPE,1mg/ml and 0.1mg/ml LBP have greater ability to inhibit apoptosis of blue light-induced damage hRPE than 0.01mg/ml LBP, the possible mechanism is that LBP can inhibit the level of mitochondrial reactive oxygen species and protect of the mitochondrial membrane potential.