中华眼底病杂志

中华眼底病杂志

精氨酸酶抑制剂对体外高糖培养的恒河猴视网膜微血管内皮细胞的保护作用

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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ArgⅠ、iNOS mRNA相对表达量升高(t=6.836、3.342),C组RF/6A细胞中ArgⅠ、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eNOS mRNA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。

Objective To investigate the effect of arginase (Arg) inhibitor N-ω-Hydroxy-L nor-Arginine (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.

关键词: 精氨酸酶/拮抗剂和抑制剂; 视网膜血管/细胞学; 内皮细胞/生理学; 细胞,培养的

Key words: Arginase/antagonists & inhibitors; Retinal Vessels/cytology; Endothelial cells/physiology; Cells, cultured

引用本文: 张惟, 姜鉴洪, 陈松, 何广辉, 杨婧, 马映雪, 陈莉, 宋建. 精氨酸酶抑制剂对体外高糖培养的恒河猴视网膜微血管内皮细胞的保护作用. 中华眼底病杂志, 2017, 33(3): 281-285. doi: 10.3760/cma.j.issn.1005-1015.2017.03.014 复制

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