摘要目的 观察精氨酸酶(Arg)抑制剂N羟基-正-L精氨酸(nor-NOHA)对体外高糖培养的恒河猴视网膜微血管内皮细胞(RF/6A细胞)的保护作用.方法 体外培养RF/6A细胞,并将其分为正常对照组(A组)、高糖对照组(B组)、高糖+nor-NOHA处理组(C组)、高糖+二甲基亚砜(DMSO)对照组(D组).A组细胞以5.5 mmol/L葡萄糖继续培养;B～D组以25.0 mmol/L葡萄糖继续培养,C、D组分别加入125 mg/L的nor-NOHA及1％DMSO.采用噻唑蓝比色法、Transwell小室法和体外成管实验分别检测各组RF/6A细胞增生、迁移和管腔形成能力.采用荧光定量聚合酶链反应(RT-PCR)检测各组RF/6A细胞中ArgⅠ、内皮型一氧化氮(NO)合酶(eNOS)、诱导型NO合酶(iNOS)的mRNA相对表达量.采用酶链免疫吸附测定试验(ELISA)检测各组RF/6A细胞培养上清液中NO和白细胞介素(IL)-1b的分泌量.结果 B组RF/6A细胞增生、凋亡及管腔形成能力较A组明显降低,差异有统计学意义(t=2.367、5.633、7.045,P＜0.05);C组RF/6A细胞增生、凋亡及管腔形成能力较B组提高,差异有统计学意义(t=5.260、6.952、8.875,P＜0.05).RT-PCR检测结果显示,与A组比较,B组RF/6A细胞中Arg Ⅰ、iNOS mRNA相对表达量升高(t=6.836、3.342),C组RF/6A细胞中Arg Ⅰ、iNOS mRNA相对表达量降低(t=4.904、7.192),差异均有统计学意义(P＜0.05);B组RF/6A细胞中eNOS mRNA相对表达量降低(t=4.165),C组RF/6A细胞中eNOSmRNA相对表达量升高(t=6.594),差异均有统计学意义(P＜0.05).ELISA检测结果显示,与A组比较,B组RF/6A细胞培养上清液中NO分泌量减少(t=4.925),C组RF/6A细胞培养上清液中NO分泌量增多(t=5.368),差异均有统计学意义(P＜0.05);B组RF/6A细胞培养上清液中IL-1b分泌量增多(t=5.032),C组RF/6A细胞培养上清液中IL-1b分泌量减少(t=7.792),差异均有统计学意义(P＜0.05).结论 nor-NOHA对体外高糖培养的RF/6A细胞有保护作用,可提高其增生、迁移及管腔形成能力;其机制可能是通过平衡Arg/NOS的表达从而抑制氧化应激反应.

abstractsObjective To investigate the effect of arginase (Arg) inhibitor N-ω-Hydroxy-L norArginine (nor-NOHA) on high glucose cultured rhesus macaque retinal vascular endothelial cell line (RF/6A) in vitro.Methods The RF/6A cells were divided into the following 4 groups:normal control group (5.0 mmol/L of glucose,group A),high glucose group (25.0 mmol/L,group B),high glucose with 125 mg/L nor-NOHA group (group C),and high glucose with 1％ DMSO group (group D).The proliferation,migration ability and angiogenic ability of RF/6A cells were measured by Methyl thiazolyl tetrazolium (MTT),transwell chamber and tube assay respectively.The express of Arg Ⅰ,eNOS,iNOS mRNA of RF/6A cells were measured by real-time polymerase chain reaction (RT-PCR),Enzyme-linked immuno sorbent assay (ELISA) was used to detect the expression of NO and interleukine (IL)-1b of RF/6A cells.Results The proliferation,migration,and tube formation ability of group A (t=2.367,5.633,7.045;P＜0.05) and group C (t=5.260,6.952,8.875;P＜0.05)were significantly higher than group B.RT-PCR results showed the Arg Ⅰ and iNOS expression in group B was higher than that in group A (t=6.836,3.342;P＜0.05) and group C (t=4.904,7.192;P＜0.05).The eNOS expression in group B was lower than that in group A and group C (t=4.165,6.594;P＜0.05).ELISA results showed NO expression in group B was lower than that in group A and group C (t=4.925,5.368;P＜0.05).IL-1b expression in group B was higher than that in group A and group C (t=5.032,7.792;P＜0.05).Conclusions Nor-NOHA has a protective effect on cultured RF/6A cells in vitro and can enhance its proliferation,migration and tube formation.The mechanism may be inhibiting the oxidative stress by balancing the expression of Arg/NOS.