The Effect Of Lycium Barbarum Polysaccharide To Senile Of Human Retinal Pigment Epithelial Cells

Objective:Age-related macular degeneration (AMD) is a serious eye disease for aged which could even lead to blind at last. This disease is highly related to the senile change of retinal pigment epithelial cells(RPE). the senile of RPE is maily embadied in three fasets: the deline of phagocytosis and the hyperplasia activity of the RPE cells ,the persistant accumulation of lipofuscin happening in RPE cells.To suspend the prograss of the senile of RPE is important to the clinical prophylaxis and treatment of AMD. Reseatched revealed that lycium barbarum polysaccharide (LBP) extracted from Barbary Wolfberry Fruit could remove oxygen free radical and lipofuscin from cells,promote cell hyperplasia activity and protect cell membranes.Our experiments aimed to establish a model of senile RPE formation, abserve the effects of different concentration lycium barbarum polysaccharide on cultured human RPE cells for phagocytosis/lipofuscin and growth, to investigate the effects of LBP to the senile of hRPE in vitro.Methods:1. Culuring retinal pigment epithelial cells and filtering suitable conce- ntration of LBP for experiment:culture hRPE-19 cell;Observing the effects of different concentration of lycium barbarum polysaccharide to the RPE cells by CCK-8, to choose the suitable concentration of lycium barbarum polysaccharide (LBP) for experiment.2. Examing the effect of lycium barbarum polysaccharide on RPE phagocytosis: Extracting photoreceter outer segment(POS) with density gradient way, mixed up with RPE to culture for different hRPE formation; Using fluorescent microscope and image analysis software to terminate effect of POS-phagocytosis of RPE fed with different concentration of LBP respectively for 12h,24h,48h, The average quantity of the ingested POS was analyzed by quantification of FITC-fluorescence emission.3. Detacting the effect of LBP on RPE lipofuscin and RPE growth: Feeding retinal pigment epithelial cells with Photoreceter outer segment for 3 weeks, modelling senile hRPE formation;then detacting hRPE lipofuscin by Flow cytometry(FCM) and transmission electron microscope(TEM).The effect of different concentration LBP on lipfuscin were tested by FCM at 12h,24h,48h.Then examing hRPE proliferation by tetrazolium salt (MTT) assay intervened in different concentration LBP after12h,24h,48h.Results:1. The human retinal pigment epithelial cells(hRPE-19 cell strain)presented longfusiform or polygon-based shape,monolayer adherence growing, every 3 to 5 days, one generation could be steadily transferred.CCK-8 indicated that lycium barbarum polysaccharide of 10ug/ml,100ug/ml,1000ug/ml had no evident influence to the hRPE cells ,while LBP that of 10ug/ml,100ug/ml were bad to the growth of hRPE cells.2. The Optiprep density gradient method could properly extrame Photoreceter outer segment, the purified POS is rhabdoid, whose size is well-propertioned.The study showed that the POS-phagocytosis by RPE cells was confirmded,and the phagocytosis of the hRPE fed with 100ug/ml and 1000ug/ml LBP were more than the group fed with no LBP.3. Transmission electron microscopy(TEM) showed that there were boviously more lipofuscin formation in RPE cells fed with POS after 3 weeks. LBP at concentrations of 10ug/ml,100ug/ml,1000ug/ml could dereased the intensity of PRE fluorescence after 12h,and the derease were isolated to dose-dependent and time-dependent manners.RPE cells with LBP at concentrations of 10ug/ml,100ug/ml,1000ug/ml had remarkable optical density differences compared with no LBP cells after 12h,24h,48h and there also were a dose-depended and time-depended manners.Conclusion:1. Our study indicated that lycium barbarum polysaccharide(LBP) with low concentration had no affect on the groth of RPE,while once the concentration become higher, the groth of RPE was contained.2. LBP with suitable concentration could promote the function of phagocytosis of RPE.3.LBP with suitable concentration could not only remove lipofuscin but also promote RPE prolifiration, which had dose-dependent and time-dependent effect to the extent.4.The function of LBP to promote hRPE cells phagocytosis,removeing lipofuscin and prolifiration indicates that LBP is good for hRPE anti-aging, which might offord a effective method for prevention and therapy for AMD.