【摘要】 目的:探究mi R-939-5p对糖尿病性视网膜病变和人视网膜微血管内皮细胞(HRMEC)的调控作用。方法:将mi R-939-5p模拟物(mi R-939-5p-mimic)或mi R-939-5p抑制剂(mi R-939-5p-inhibitor)转染到HRMEC中,并将细胞用高糖(HG组,25 m M)或低糖(LG组,5 m M)处理24 h。通过细胞计数试剂盒8(CCK-8)来检测细胞活力,EdU法检测细胞DNA的复制能力,Hoechst 33258染色检测细胞凋亡,使用双荧光素酶试剂盒E2920验证mi R-939-5p与NOS2 3’-UTR之间的结合关系。对大鼠腹腔注射65 mg/kg的链脲佐菌素(STZ)诱导DR模型,通过RT-qPCR检测mi R-939-5p水平,Western Blot检测诱导型一氧化氮合酶(NOS2)水平,苏木精伊红(HE)染色检查大鼠视网膜形态,免疫组织化学染色检测视网膜Claudin-5和Occludin的表达,伊文思蓝染色检测大鼠血视网膜屏障(BRB)通透性,ELISA法检测大鼠房水中白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的水平。结果:与LG组的HRMEC相比,HG组的mi R-939-5p显著降低,而NOS2蛋白水平显著升高(P<0.05)。荧光素酶活性测定显示,与NC-mimic组相比,mi R-939-5p-mimic与pGL3-NOS2-WT共转染组的荧光素酶活性显著降低(P<0.05)。与HG+NC-mimic组相比,HG+mi R-939-5p-mimic组的mi R-939-5p水平和细胞活力显著升高,而NOS2蛋白水平和细胞凋亡率显著降低(P<0.05)。与DR组相比,mi R-939-5p-Agomir组大鼠视网膜组织病变减轻,Claudin-5和Occludin的表达水平明显升高,伊文思蓝浓度显著降低(P<0.05);与DR组相比,mi R-939-5p-Agomir组大鼠房水中IL-1β和TNF-α的水平均显著降低(P<0.05)。结论:在高糖培养的HRMEC中和DR大鼠视网膜中,mi R-939-5p为低表达模式,NOS2为高表达模式。上调mi R-939-5p通过靶向抑制NOS2对DR大鼠视网膜和HRMEC提供保护作用。更多还原

【Abstract】 Objective: To investigate the regulatory effect of mi R-939-5 p on diabetic retinopathy(DR) and human retinal microvascular endothelial cells(HRMEC). Methods: mi R-939-5 p-mimic or mi R-939-5 p-inhibitor was transfected into HRMEC, and the cells were treated with high glucose(HG, 25 mM) or low glucose(LG, 5 mM) for 24 hours. Cell viability was detected by cell counting kit8(CCK-8), DNA replication ability was detected by EdU method, and apoptosis was detected by Hoechst33258 staining. The binding relationship between mi R-939-5 p and NOS2 3’-UTR was verified by double luciferase kit E2920. The rat model of DR was induced by intraperitoneal injection of streptozotocin(STZ) of 65 mg/kg. The level of mi R-939-5 p was detected by RT-qPCR, the level of inducible nitric oxide synthase(NOS2) was detected by Western Blot, the morphology of rat retina was examined by hematoxylin-eosin(HE) staining, the expression of Claudin-5 and Occludin was detected by immunohistochemical staining, and the permeability of the blood retinal barrier(BRB) in rats was detected by Evans blue staining. The levels of interleukin-1 β(IL-1 β) and tumor necrosis factor-α(TNF-α) in aqueous humor of rats were detected by ELISA. Results: Compared with HRMEC in LG group, mi R-939-5 p in HG group was significantly reduced, while NOS2 protein level was significantly increased(P<0.05). The luciferase activity measurement showed that, compared with NC-mimic group, the luciferase activity of the mi R-939-5 p-mimic and pGL3-NOS2-WT co-transfected group was significantly reduced(P<0.05). Compared with HG+NC-mimic group, the mi R-939-5 p level and cell viability of HG+mi R-939-5 p-mimic group were significantly increased, while the NOS2 protein level and the apoptosis rate were significantly reduced(P<0.05). Compared with DR group, rats in mi R-939-5 p-Agomir group had less retinal tissue lesions, the expression levels of Claudin-5 and Occludin were significantly increased, and the concentration of Evans blue was significantly reduced(P<0.05). Compared with DR group, the levels of IL-1β and TNF-α in the aqueous humor of rats in mi R-939-5 p-Agomir group were significantly reduced(P<0.05). Conclusion: In the high glucose cultured HRMEC and DR rat retina, mi R-939-5 p is a low expression pattern, and NOS2 is a high expression pattern. Up-regulation of mi R-939-5 p provides a protective effect on the retina and HRMEC of DR rats by targeting NOS2.更多还原