枸杞多糖对人视网膜色素上皮细胞抗衰老作用的影响

The anti-aging effect of lycium barbarum polysaccharide on human retinal pigment epithelial cell

背景 年龄相关性黄斑变性（AMD）的发病与视网膜色素上皮（RPE）细胞的衰老有关,寻求抗RPE细胞衰老的药物对AMD的防治有重要意义.研究表明枸杞多糖（LBP）有延缓衰老的作用,但其对RPE细胞衰老的作用机制尚不完全明确. 目的 研究LBP对体外培养的人RPE细胞的吞噬功能、清除脂褐素的功能及细胞增生活性的影响,探讨LBP防治AMD的可行性.方法 分离猪视网膜神经上皮层,密度梯度离心法提取猪眼光感受器外节（POS）,并用异硫氰酸荧光素（FITC）标记,然后分别加入含0.01、0.10、1.00 g/LLBP的人RPE中培养24 h,荧光显微镜下测量正常对照组、POS对照组及各质量浓度LBP＋POS组人RPE细胞吞噬POS的荧光面积,评估LBP对人RPE细胞吞噬功能的影响.将密度为5×104个/ml的人RPE细胞与猪POS混合培养3周以建立POS诱导的脂褐素模型,然后将模型细胞分别加入含上述质量浓度LBP的培养液中,用流式细胞术定量检测人RPE细胞内脂褐素的自发荧光值（A）,观察LBP对人RPE细胞清除脂褐素功能的影响.对脂褐素化的人RPE细胞进行培养后分别加入含有上述不同质量浓度LBP的培养液,采用细胞计数试剂盒-8（CCK-8）法测定培养24、48和72 h后各组细胞生长的增生值（A）. 结果 光学显微镜下可见猪POS呈大小均一、排列整齐、独立的细杆状形态,透射电子显微镜下可见双层膜盘样结构,荧光显微镜下可见FITC标记后呈黄绿色荧光.正常对照组未发现人RPE细胞对猪POS的吞噬,但随着LBP质量浓度的增加,RPE细胞对猪POS的吞噬面积逐渐增加,差异有统计学意义（F=21.425,P=0.006）.与POS对照组比较,不同质量浓度LBP＋POS组人RPE细胞对猪POS的吞噬面积均增加,差异均有统计学意义（P＜0.01）.流式细胞术检测结果表明,正常对照组RPE细胞中脂褐素荧光值非常低,RPE细胞与POS共培养后细胞中脂褐素荧光值明显增加,不同质量浓度的LBP作用后,RPE细胞内脂褐素自发荧光值逐渐下降,均低于POS对照组,差异均有统计学意义（P＜0.01）,其中1.00 g/L LBP＋POS组RPE细胞内脂褐素自发荧光值最低,LBP对脂褐素的清除能力最强.LBP干预人RPE细胞24、48、72 h,正常RPE细胞组的细胞增生值（A）均最高,脂褐素化RPE细胞组A值最低,随着LBP质量浓度的增加,各质量浓度LBP＋脂褐素化RPE细胞组A值逐渐增加,各时间点1.00 g/L LBP＋脂褐素化RPE组A值接近正常RPE细胞组,差异均无统计学意义（P＞0.05）.结论 LBP能增强人RPE细胞的抗衰老作用,其作用机制与增强人RPE细胞吞噬POS、清除脂褐素的功能及细胞增生活性有关.

Background The pathogenesiof age-related maculadegeneration （AMD） iassociated with the senility of human retinal pigmenepithelium （RPE） cells.Seeking drug to arresRPE cell senility iof significance fothe prevention and treatmenof AMD.Research showed thathe lycium barbarum polysaccharide （LBP） can delay senility,buitinfluence on RPE cell aging iunclear.Objective Thistudy wato discusthe protective effecand mechanism of LBP on RPE cell aging.MethodPorcine retinal neural epithelial layewaisolated,and photoreceptooutesegmen（POS） waextracted by density gradiencentrifugation and marked by FITC.The POwathen co-cultured with RPE cellin the medium containing 0.01,0.10 and 1.00 g/L LBP fo24 hours.The areof fluorescence,representing the amounof POphagocytosed by RPE cells,wameasured undethe fluorescenmicroscope to evaluate the influence of LBP on the phagocytifunction of RPE cells.The POS-induced RPE lipofuscin-uptake cell model waestablished by co-culturing human RPE cellwith porcine POfo3 weeks.The RPE-POco-culture cell model waincubated in medium containing 0.01,0.10 o1.00 g/L LBP,and the autofluorescence caused by lipofuscin up-taken into RPE cellwadetected with flow cytometry.cell counting kiwaused to assescell proliferation and viability （value） 24,48 and 72 hourafteculturing.ResultPorcine POpresented athin rodundethe lighmicroscope and appeared abilayedisc-like structureundethe transmission electron microscope,and itFITC-labeled yellow-green autofluorescence waobserved undethe fluorescenmicroscope.No POwaup-taken into the RPE cellin the normal control group,buthe areof POphagocytosed by RPE cellwagradually enlarged with increasing doseof LBP,showing significandifference among the group（F =21.425,P =0.006）.Compared with the POcontrol group,the phagocytosed areincreased avariouconcentrationof LBP＋POgroup（P〈0.01）.Flow cytometry showed thathe autofluorescence value in the POcontrol group wamore highethan thaof the normal control group.Athe LBP dose increased,the autofluorescence value in the RPE celldeclined gradually and iwaneathe normal value in the 1.00 g/L LBP＋ POgroup.The rate of proliferation of the lipofuscin RPE cellvaried with the increase of doseof LBP with the maximal value in the normal RPE group and minimal value in the lipofuscin RPE group,and the rate of proliferation of the lipofuscin RPE cellascended with increasing doseof LBP until neathe normal value in the 1.00 g/L LBP ＋ lipofuscin RPE cellgroup （P〉0.05）.ConclusionLBP enhance the anti-aging effecof human RPE cellby strengthening the phagocytiability to POand the ability to remove lipofuscin and by heightening the proliferation of human RPE cells.