摘要背景 近年来糖尿病视网膜病变(DR)的研究聚焦于药物治疗.促红细胞生成素(EPO)能减少DR神经组织的结构和功能损伤,但具有促进视网膜新生血管形成的潜在危险.氨甲酰EPO(CEPO)具有相同的神经保护作用,且无促进新生血管形成的作用,但其神经保护作用尚未在眼部疾病,尤其是DR中得到证实.目的 比较CEPO与EPO在抗DR的神经成分和血管成分中的作用及机制.方法 用化学合成法制备EPO衍生物CEPO.将60只清洁级雄性SD大鼠按随机数字表法分为正常对照组、DM组、DM+ EPO组和DM+CEPO组,用链脲佐菌素(STZ)60 mg/kg腹腔内注射建立大鼠DM模型,而正常对照组大鼠腹腔内注射等体积枸橼酸-枸橼酸钠缓冲液.每周监测大鼠血糖.造模后4周,DM+EPO组和DM+CEPO组大鼠分别腹腔内注射50 μg/kg EPO和CEPO,正常对照组和DM组大鼠均采用等量双蒸水腹腔内注射.干预后2周对各组大鼠进行视网膜电图(ERG)检查,然后处死大鼠摘除眼球制备视网膜标本,分别进行视网膜组织学检查,采用TUNEL法检测视网膜神经节细胞(RGCs)凋亡情况,采用实时荧光定量PCR和Western blot法分别检测各组大鼠视网膜中异源二聚体受体(CD131)、EPO受体(EPO-R)、胸腺细胞分化抗原-1(Thy-1)、胶质纤维酸性蛋白(GFAP)和血管内皮生长因子(VEGF) mRNA及其蛋白质的表达.结果 合成产物为纯度较高的CEPO.造模后至药物干预后2周,DM组、DM+EPO组和DM+CEPO组大鼠体质量明显低于正常对照组,而血糖水平明显高于正常对照组,4个组间差异均有统计学意义(F=49.39、455.91,均P=0.00);DM组大鼠ERG OPs振幅较正常对照组大鼠明显下降,差异有统计学意义(P=0.03),而DM+ EPO组与DM+CEPO组OPs波振幅与正常对照组大鼠比较差异均无统计学意义(P=0.55、0.49);4个组间大鼠ERG a波、b波振幅的差异均无统计学意义(F=0.30、1.12,P＞0.05).与正常对照组大鼠比较,DM组大鼠视网膜组织厚度变薄,RGCs计数减少,视网膜中TUNEL染色阳性细胞增多,而DM+ EPO组和DM+CEPO组大鼠视网膜全层厚度、内丛状层(IPL)、内核层(INL)厚度均明显薄于DM组大鼠,4个组间差异均有统计学意义(F=61.23、23.35、13.33,均P=0.00),RGCs数目明显多于DM组,4个组间差异有统计学意义(F=15.64,P=0.00).DM组大鼠视网膜中Thy-1 mRNA(2-ΔΔCt)及其蛋白表达量(A值)明显低于正常对照组,DM+EPO组和DM+CEPO组大鼠视网膜中Thy-1 mRNA及其蛋白表达量明显高于DM组,差异均有统计学意义(P＜0.05).DM组大鼠视网膜中GFAP mRNA(2-ΔΔCt)及其蛋白表达量(A值)明显升高于正常对照组大鼠,DM+EPO组和DM+CEPO组大鼠视网膜中GFAP mRNA及其蛋白表达量明显低于DM组,差异均有统计学意义(P＜0.05).DM+CEPO和DM+EPO组间大鼠视网膜Thy-1或GFAP表达量差异均无统计学意义(P＞0.05).DM+EPO组大鼠视网膜中EPOR mRNA及其蛋白表达量明显高于其他各组,而DM+CEPO组大鼠视网膜中CD131 mRNA及其蛋白表达量明显升高,差异均有统计学意义(P＜0.05).在DM组和DM+EPO组大鼠VEGF mRNA及其蛋白表达量均明显高于正常对照组大鼠,DM+CEPO组大鼠视网膜中VEGF mRNA及其蛋白表达量明显低于DM+EPO组,差异均有统计学意义(P＜0.05).结论 CEPO在防治DR患者视网膜神经细胞损伤中的作用与EPO相似,且不存在EPO的促新生血管形成的作用.CEPO可能通过CD131受体发挥视网膜神经保护作用.

abstractsBackground Recently the study on diabetic retinopathy (DR) primarily fucoses on the medication research.Erythropoietin (EPO) has the neuroprotective role on retinal neural cells in DR,but it is an important vasculogenesis growth factor.Carbamylated erythropoietin (CEPO) is proved to have the similar neuroprotection to EPO,what is more,CEPO have no the vasculogenesis effect.However,the neural protection of CEPO has not been verified in DR.Objecctive This study was to investigate the effects of CEPO on DR and compare the differences of protective effects between CEPO and EPO in DR and further to elucidate the underlying mechanism.Methods CEPO,the derivation of EPO,was synthesized by chemical synthesis and evaluated by chromatography,enzyme digestion,SDS-PAGE electrophoresis and silver nitrate dye.Sixty eight-week-old clean male Sprague-Dawley rats were assigned to four groups according to the random number table.Diabetic mellitus (DM) models were established by intraperitoneal injection of 60 mg/kg streptozotocin (STZ) in the rats of the DM group,DM+EPO group and DM+CEPO group,and equivate volume of citric acid-sodium citrate buffer solution was used in the same way in the normal control group.Four weeks later,50 μg/kg EPO or CEPO solution was intraperitoneal injection in the rats of the DM +EPO group and DM+CEPO group,respectively,and retinal examinations were performed 2 weeks after intervention.Retinal founction was evaluated by electroretinogran (ERG),and then the rats were sacrificed.Retinal samples were prepared for the histopathological examination.Retinal gangline cells (RGCs) apoptosis was detected by TUNEL assay.Real-time PCR and Western blot were used to assyed the expressions of betacommon receptor (CD131),EPO receptor (EPO-R),Thy-1,glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) in retina in protein and genetic levels.Results Purified CEPO was obtained by chemical synthesis.The body wight was significantly lower and the blood glucose level was higher in the rats of the DM group,DM+EPO group and DM +CEPO group than that in the normal control group 2 weeks after modeling,with significant difference among the four groups (F =49.39,455.91,all at P =0.00).No significant differences were found in a amplitude and b amplitude among the groups(F=0.30,1.12,both at P＞0.05),but the oscillatory potentials (OPs) amplitude was signicanntly reduced in the DM group compared to the normal control group (P=0.03),but the OPs amplitudes were not significantly different between DM + EPO group and DM + CEPO group compared with the normal control group (P =0.55,0.49).Compared with the normal control group,retinas were thiner and RGCs were decreased,and the positive cells for TUNEL assay were significantly increased in the DM group.However,the thicknesses of entire retinas,inner plexiform layer and inner nuclear layer were reduced in comparison with the normal control group,with significant differences among the groups (F=61.23,23.35,13.33,all at P=0.00),but the number of RGCs was significantly larger in the DM+EPO group and DM+CEPO group compared with DM group (F=15.64,P =0.00).The expressions of Thy-1 mRNA (2 ΔΔCt) and protein (A value) in DM group,and the expressing levels were increseased in the DM +EPO group and DM +CEPO group (P＜0.05).The expressions of GFAP mRNA (2-ΔΔCt) and its protein (A) were significantly downregulated in the DM+EPO group and DM+CEPO group compared with DM group (P＜0.05).The EPOR mRNA and protein expressions in the retinas showed the highest levels in DM+EPO group,and those of CD131 mRNA and protein were highest in DM +CEPO group (P＜0.05).The VEGF expressions in the protein and genetic levels were significantly higher in DM group and DM+EPO group that those of DM+CEPO group,showing significant differences among the groups (all at P＜0.05).Conclusions CEPO shows a similar protective effect on retinal neurons in DR without promorting angiogenesis.CEPO might exert its neuroprotective effect through CD131 receptor,which is different from EPO-EPOR mechanism.