摘要背景 抑制视网膜新生血管形成是目前相关眼病治疗的主要目标之一,而寻找视网膜新生血管形成过程中的干预靶点是研究的热点.研究表明,胰岛素样生长因子1(IGF-1)可促进血管内皮细胞增生及抑制血管内皮细胞凋亡,但其是否可以作为视网膜新生血管类眼病的治疗靶点尚不清楚. 目的 探讨IGF-1对体外培养的人视网膜血管内皮细胞(HRECs)的迁移、凋亡和管腔形成能力的作用及其机制.方法 对HRECs进行体外培养,取对数生长期的细胞进行实验.采用逆转录PCR法检测培养细胞中IGF-1受体(IGF-1R)mRNA的表达.按照检测指标的不同分别于培养液中添加不同质量浓度IGF-1溶液培养细胞,待细胞生长至约90％融合后用无菌移液枪头垂直于培养板底划痕,用Photoshop CS4软件检测、计算和比较0、10和200 ng/ml IGF-1组划痕后12 h和24 h与划痕前HRECs迁移的面积差(试验前面积-试验后面积,△S);采用流式细胞术测定并比较0 ng/ml IGF-1组与1 000 ng/ml IGF-1组细胞培养后24 h HRECs凋亡比例的差异;采用Matrigel体外三维成型法检测并比较0、10、100和200 ng/ml IGF-1组细胞培养后24 h完整管腔的形成数量;采用实时荧光定量PCR法检测并比较0、500及1 000 ng/ml IGF-1组细胞培养后6 h HRECs中血小板衍生因子(PDGF)-BB和caspase-3 mRNA的相对表达量. 结果 HRECs培养后2～3d达90％以上融合,细胞排列紧密,琼脂糖凝胶电泳结果显示IGF-1R mRNA呈阳性表达.划痕后12 h和24 h,各组HRECs的△S随着IGF-1质量浓度的增加而逐渐增加,划痕后24h,200 ng/ml IGF-1组HRECs的△S为(4.83±0.61) ×105 μm2,较0 ng/ml IGF-1组的(3.28±0.64) ×105 μm2明显增大,差异有统计学意义(t=-3.707,P=0.021).0 ng/mlIGF-1组与1 000 ng/ml IGF-1组凋亡细胞比例分别为(18.77±2.37)％和(12.05±0.88)％,差异有统计学意义(t=2.869,P=0.046).200 ng/ml IGF-1组HRECs完整管腔形成数量为(20.33±2.83)个/孔,数目高于0 ng/ml IGF-1组的(17.94±1.96)个/孔,差异有统计学意义(t=-2.940,P=0.042).与0 ng/ml IGF-1组比较,1 000 ng/ml IGF-1组细胞中PDGF-BB mRNA的相对表达量明显升高,而caspase-3 mRNA的相对表达量明显降低,差异均有统计学意义(t=-3489,P=0.025;t=7.287,P=0.002).结论 IGF-1促进体外培养HRECs的迁移和血管管腔形成,抑制HRECs凋亡,其作用机制可能与上调HRECs中PDGF-BB的表达及下调caspase-3的表达有关.

abstractsBackground The suppression of retinal angiogenesis is one of primary treatment targets for retinal vascular diseases,so seeking the intervention targets of retinal neovascularization is a hot research.Studies showed that insulin-like growth factor 1 (IGF-1) can promote the growth and restrain the apoptosis of vascular endothelial cells.However,whether IGF-1 is an intervention target for the treatment of retinal vascular diseases is unelucidated.Objective This study was to address the effects of IGF-1 on the migration,apoptosis and capillary tube formation of human retinal vascular endothelial cells (HRECs) and mechanism.Methods HRECs were cultured in vitro,and the cells in the exponential phase were prepared for subsequent experiments.The expression of IGF-1R mRNA in the cells was examined using reverse transcriptase PCR assay.Different concentrations of IGF-1 were added in the medium based on the difference of tests.The relative free-cell area difference (△S) after test was measured by Photoshop CS4 software and compared among 0,10 and 200 ng/ml IGF-1 groups 12 and 24 hours after cell scratching,respectively.The cell apoptotic rate was assayed by flow cytometry and compared between 0 ng/ml IGF-1 group and 1 000 ng/ml IGF-1 group,and the number of capillary tubes was examined by Matrigel test and assessed among 0,10,100 and 200 ng/ml IGF-1 groups 24 hours after addition of IGF-1.The expressions of platelet derived growth factor (PDGF)-BB mRNA and caspase-3 mRNA in the cells of the 0,500 and 1 000 ng/ml IGF-1 groups were detected by real-time fluorescence quantitative PCR after adding IGF-1 for 6 hours.Results Cultured cells grew well and attached 90％ confluence 2-3 days after incubation,and IGF-1R mRNA was positively expressed in the cells.In 12 and 24 hours after scratching,the relative migrating area of the cells was gradually reduced with the increase of IGF-1 contents.The △S was (4.83 ± 0.61) × 105 μm2 in the 200 ng/ml IGF-1 group,which was significantly larger than (3.28±0.64) ×105 μm2 in the 0 ng/ml IGF-1 group 24 hours after stretching (t=-3.707,P=0.021).The apoptotic rate in the 0 ng/ml IGF-1 group and 1 000 ng/ml IGF-1 group was (18.77±2.37) ％ and (12.05 ±0.88) ％,with a significant difference between them (t =2.869,P =0.046).The number of intact tubes was significantly increased in the 200 ng/ml IGF-1 group compared with the 0 ng/ml IGF-1 group ([20.33±2.83]/well vs.[17.94± 1.96]/well;t =-2.940,P =0.042).Compared with 0 ng/ml IGF-1 group,the relative expression level of PDGF-BB mRNA was elevated and that caspase-3 mRNA was evidently reduced in the 1 000 ng/ml IGF-1 group (t=-3.489,P =0.025;t =7.287,P =0.002).Conclusions IGF-1 can promote the migration and angiogenesis of HRECs and inhibit the apoptosis of HRECs.These effects of IGF-1 probably are associated with the up-regulation of PDGF-BB and down-regulation of caspase-3 in the cells.