Background Researches showed that ADP-ribosylation factor (ARF) promotes intracorneal secretion of multiple angiogenesis-related factors,such as vascular endothelial growth factor (VEGF) and nitricoxide synthase (NOS) etc., and therefore results in corneal neovascularization.However, whether ARF affects the tube formation of human retinal endothelial cells(HRECs) is unelucidated.Understanding the effect of ARF tube formation of HRECs is important for the target treatment of retinal vascular diseases.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on tube formation of HRECs in vitro.Methods HRECs (HREC line) were cultured and passaged.The growth-well cells were harvested and divided into two groups.The cells were regularly cultured in the control group,and ARF antagonist (lml) was added in the culture medium in the ARF antagonist group.The expression levels of ARFmRNA and protein in the cells were examined by reverse transcription (RT)-PCR and Western blot.The morphology and number of HREC tube formation were detected by using three-dimensional Matrigel assay.The relative expression levels of VEGF, NOS, focal adhesion kinase (FAK) and heat shock protein 90 (HSP90) at gene level and protein level were examined by RT-PCR and Western blot in vitro.Results The relative expression levels of ARFmRNA in the cells were 0.65 ±0.14 and 0.32±0.10, and those of ARF protein were 0.85±0.15 and 0.24±0.17 in the control group and ARF antagonist group,showing significant differnces between the two groups (t =7.32, P =0.00;t =5.15, P =0.00).The number of HREC tube formation was (34.66±8.57)/field in the ARF antagonist group, which was significantly lower than (51.46±7.12)/field in the control group (t=2.99 ,P=0.04).The relative expression levels of VEGF mRNA, NOSmRNA and their proteins in the cells were significantly lower than those of the control group (t =3.02, P =0.04;t =3.68, P =0.02;t =3.33,P=0.03;t=2.89 ,P=0.04).The relative expression levels of FAKmRNA and HSP90mRNA in the ARF antagonist group were 0.65±0.18 and 0.28±0.05 ,which were significantly lower than 0.76±0.25 and 0.46±0.09 in the control group (all at P＜0.05).Conclusions ARF antagonist appears to have an inhibitory effect on the tube formation ability of HRECs propably by down-regulating the expressions of VEGF, NOS and the downstream signal transduction factors FAK and HSP90 in HRECs in vitro.%背景 研究证实ADP-核糖基化因子(ARF)能促进角膜组织中促血管相关因子的分泌,如血管内皮生长因子(VEGF)和一氧化氮合酶(NOS)等,从而导致病理状态下角膜新生血管的发生,但ARF对人视网膜膜血管内皮细胞(HRECs)管腔形成能力的影响和机制尚不清楚,了解ARF对HRECs形成管腔能力的影响对研究视网膜新生血管相关疾病的靶向治疗具有重要意义.目的 探讨ARF拮抗剂对HRECs管腔形成能力的影响以及其作用机制.方法 对HRECs进行常规培养和传代,取对数生长期细胞分为对照组和ARF拮抗剂组,对照组细胞在实验过程中按常规培养法培养,培养基中不添加ARF拮抗剂,而ARF拮抗剂组细胞在后续实验时于培养液中添加15 μmol/L的ARF拮抗剂1 ml.采用逆转录PCR法和Western blot法检测ARF mRNA及蛋白在HRECs中的表达.应用半固态Matrigel胶体外三维成型法检测对照组和ARF拮抗剂组HRECs形成管腔形态和数目.采用RT-PCR和Western blot法检测VEGF、NOS、局部黏着斑激酶(FAK)、热休克蛋白90(HSP90) mRNA及蛋白在各组HRECs中的相对表达量.结果 逆转录PCR和Western blot法检测显示,对照组和ARF拮抗剂组HRECs中ARF mRNA的相对表达量分别为0.65士0.14和0.32±0.10,对照组和ARF拮抗剂组HRECs中ARF蛋白的相对表达量分别为0.85±0.15和0.24±0.17,ARF拮抗剂组ARFmRNA及其蛋白的相对表达量均明显低于对照组,差异均有统计学意义(t=7.32,P=0.00;t=5.15,P=0.00).对照组和ARF拮抗剂组HRECs形成管腔的数量分别为(51.46±7.12)和(34.66±8.57)个/视野,差异有统计学意义(t=2.99,P=0.04).RT-PCR和Western blot法检测显示,ARF拮抗剂组HRECs中VEGFmRNA和NOS mRNA及蛋白的相对表达量均明显低于对照组,差异均有统计学意义(t=3.02,P=0.04;t=3.68,P=0.02;t=3.33,P=0.03;t=2.89,P=0.04).ARF拮抗剂组HRECs中FAK mRNA和HSP90 mRNA的相对表达量分别为0.65±0.18和0.28±0.05,均明显低于对照组中0.76±0.25和0.46±0.09,差异均有统计学意义(均P＜0.05).结论 ARF拮抗剂对HRECs的管腔形成能力有抑制作用,其作用机制可能与下调VEGF、NOS和下游重要信号转导因子FAK、HSP90在HRECs中的表达有关.
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