【摘要】 背景:有实验表明,促红细胞生成素对视网膜的光化学损伤有保护作用,该作用与光化学损伤视网膜色素上皮细胞中半胱天冬酶-3表达的变化有关?目的:采用光刺激诱导人视网膜色素上皮细胞凋亡,观察不同剂量促红细胞生成素对细胞半胱天冬酶-3表达的影响。设计:观察对比实验。单位:青岛大学医学院。材料:成人视网膜色素上皮-19细胞株购自美国细胞培养收集公司。DMEM/F12混合培养基、新生牛血清、胰蛋白酶购自GIBCO生物技术公司。重组人促红细胞生成素购自Sigma生物技术公司。人半胱天冬酶-3定量EIA试剂盒购自上海西唐生物科技有限公司。半胱天冬酶-3单克隆抗体购自美国Santa Cruz公司;PV6001免疫组织化学试剂盒和DAB显色试剂盒购自北京中山生物技术公司。方法:实验于2006-05/2007-01在青岛大学医学院病理生理教研室完成。取成人视网膜色素上皮-19细胞株传代培养的2～5代细胞建立光损伤模型,传代细胞随机分为7组,每组4孔,①正常对照组:不加光照,不加促红细胞生成素药物干预。②光损伤模型组:光照12h,不加促红细胞生成素药物干预。③光损伤+10000 U/L促红细胞生成素组、光损伤+20000 U/L促红细胞生成素组及光损伤+40000 U/L促红细胞生成素组:光照12h,分别加10000、20000及40000 U/L促红细胞生成素。④光损伤+40000 U/L促红细胞生成素组+AG490组:光照12h,加促红细胞生成素40000 U/L及Jak2激酶抑制剂50000 U/L。⑤光损伤+40000 U/L促红细胞生成素组+蛋白激酶B特异性抑制剂组:光照12h,加促红细胞生成素40000 U/L及蛋白激酶B特异性抑制剂100μmol/L。主要观察指标:采用酶联免疫吸附实验和免疫组织化学法定量检测不同含量促红细胞生成素干预治疗前后视网膜色素上皮细胞半胱天冬酶-3表达的变化。结果:正常对照组半胱天冬酶-3无明显表达;光损伤模型组半胱天冬酶-3表达于视网膜色素上皮细胞核上,呈大量特异性黄色着色;光损伤不同剂量促红细胞生成素组半胱天冬酶-3表达的特异性黄色着色随促红细胞生成素含量增加而减弱,以光损伤+40000 U/L促红细胞生成素组最弱光损伤+40000 U/L促红细胞生成素组+AG490组半胱天冬酶-3呈强阳性表达,光损伤+40000 U/L促红细胞生成素组+蛋白激酶B特异性抑制剂组半胱天冬酶-3表达仍呈阳性,但较光损伤模型组稍弱。结论:重组人促红细胞生成素可减少视网膜色素上皮细胞的光损伤后半胱天冬酶-3的表达。重组人促红细胞生成素抗光损伤诱导的视网膜色素上皮细胞凋亡作用机制之一可能是抑制半胱天冬酶-3的表达。更多还原

【Abstract】 BACKGROUND:Recent studies demonstrate that erythropoietin(EPO) can protect retina from light injury,and is the mechanism related to the expression of Caspase-3 in the light-injured retinal pigment epithelium(RPE) cells. OBJECTIVE:To study the effects of EPO at different dosages on the expression of Caspase-3 in light-injured human RPE cells. DESIGN:Control observation. SETTING:Qingdao University Medical College. MATERIALS:Adult ARPE-19 cells(American Cell Culture Collection Company);DMEM/F12 mixed medium,fetal bovine serum and trypase(GIBCO Biotechnique Company);recombinant human EPO(rhEPO,Sigma Biotechnique Company);human Caspase-3 quantitative kit(Shanghai Xitang Biotechnique Co.,Ltd);Caspase-3 monoclonal antibody(American Santa Cruz Company);PV6001 immunohistochemistry kit and DAB color reagent kit(Zhongshan Biotechnology Company,Beijing). METHODS:The experiment was carried out in the Department of Pathophysiology at Qingdao University Medical College between May 2006 and January 2007. Human RPE-19 cell strain at passages 2–5 were harvested for light injury models,and the passage cells were divided into 7 groups randomly,with 4 apertures in each group:①normal control group:no light or EPO intervention;②light-injured model group:12-hour illumination,no EPO intervention;③light-injury and EPO groups:12-hour illumination with 10 000,20 000 and 40 000 U/L EPO;④light-injury and 40 000 U/L EPO and AG490 group:12-hour illumination with 40 000 U/L EPO and inhibitor of Jak2 enzyme 50 000 U/L;⑤light-injury and 40 000 U/L EPO and carbxyl-terminal modulator protein(CTMP) group:12-hour illumination with 40 000 U/L EPO and specific inhibitor of protein kinase B enzyme 100 μmol/L. MAIN OUTCOME MEASURES:The enzyme linked immunosorbant assay(ELISA) and immunohistochemistry were used to assess the effects of rhEPO at the different doses on the expression of Caspase-3 in light-injured human RPE cells. RESULTS:Caspases-3 was not expressed in RPE cells of the normal control group and was positively expressed in the nucleus of RPE cells of the light-injured model group,showing a specific brown-yellow staining. Expression of Caspase-3 was gradually decreased in every rhEPO group with increase of EPO concentration,with the weakest expression in 40 000 U/L rhEPO group. The effects of EPO on Caspase-3 expression were strongly inhibited in light-injury+ 40 000 U/L EPO +AG490 group and the expression was positive in light-injury +40 000 U/L EPO+CTMP group,which was slightly weaker than light-injured model group. CONCLUSION:The rhEPO can reduce the expression of Caspase-3 in the light-injured human RPE cells,and one of the possible mechanisms is the inhibition of light-injured RPE cell apoptosis by the rhEPO.更多还原