NDRG1基因在体外对视网膜血管内皮细胞血管形成能力的影响  

The effect of NDRG1 gene on the angiogenesis ability of retinal endothelial cells in vitro

作　　者：曹靖靖 李辉 寇振宇 吴桂佳 东莉洁 焦明菲 Cao Jingjing;Li Hui;Kou Zhenyu;Wu Guijia;Dong Lijie;Jiao Mingfei(Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin Branch of National Clinical Research Center for Ocular Disease,Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院,眼视光学院,眼科研究所,国家眼耳鼻喉疾病临床医学研究中心天津市分中心,天津市视网膜功能与疾病重点实验室,天津300384

出　　处：《中华眼底病杂志》2024年第7期538-544,共7页Chinese Journal of Ocular Fundus Diseases

基　　金：天津市高等教育委员会科技发展基金(2022ZD057);天津市滨海新区卫生健康委员会科技项目(2022BWKZ003);天津市视网膜功能与疾病重点实验室开放项目(2021tjswmm002);天津市卫生健康科研项目(TJWJ2023ZD002);天津市医学重点学科(专科)建设项目(TJYXZDXK-037A)。

摘　　要：目的观察高糖状态下NDRG1对恒河猴视网膜血管内皮细胞(RF/6A细胞)增殖、迁移和管腔形成能力的影响。方法将RF/6A细胞分为正常组、甘露醇组、高糖组、无靶基因的小干扰RNA(siRNA)阴性对照组(siRNA组)、30 nmol/L siRNA下调NDRG1基因组(siNDRG1组)、50 nmol/L siNDRG1组。正常组细胞常规培养;甘露醇组细胞加入25 mmol/L甘露醇培养;高糖组细胞加入25 mmol/L葡萄糖培养;siRNA组细胞加入25 mmol/L葡萄糖,再加入空白siRNA诱导培养;30、50 nmol/L siNDRG1组细胞均加入25 mmol/L葡萄糖,再分别用30、50 nmol/L siNDRG1进行诱导。所有细胞均孵育24 h进行后续实验。4',6-二脒基-2-苯基吲哚染色观察细胞增殖;细胞计数试剂盒8染色检测细胞活性;实时定量聚合酶链反应、蛋白质免疫印迹法检测细胞中NDRG1基因的mRNA和蛋白表达水平;细胞划痕实验观察细胞迁移;细胞管腔形成实验检测管腔形成。两组间比较采用双尾Student t检验;多组间比较采用单因素方差分析。结果高糖组与正常组、甘露醇组细胞增殖率(t=36.659、57.645)、迁移率(t=24.745、33.638)、管腔形成数量(t=41.276、22.867)比较,差异均有统计学意义(P<0.01)。与正常组、甘露醇组比较,高糖组细胞中NDRG1基因的mRNA和蛋白相对表达量显著降低,差异有统计学意义(t=46.145、21.541、36.738、32.976,P<0.001)。与siRNA组比较,30 nmol/L siNDRG1组、50 nmol/L siNDRG1组细胞中NDRG1基因的mRNA和蛋白相对表达量明显降低,差异有统计学意义(t=44.275、40.7577、57.167、25.877,P<0.01)。与正常组、siRNA组比较,30 nmol/L siNDRG1组细胞迁移率增加,差异有统计学意义(t=57.562、49.522,P<0.01)。与正常组、siRNA组比较,30 nmol/L siNDRG1组相同视野下细胞管腔形成数量明显增加,差异有统计学意义(t=63.446、42.742,P<0.01)。结论下调NDRG1基因可诱导高糖状态下RF/6A细胞活性、细胞迁移和管腔形成能力的提高。Objective To observe the effects of NDRG1 on proliferation,migration and lumen formation of retinal vascular endothelial cells(RF/6A cells)in monkeys under high glucose condition.Methods RF/6A cells were divided into normal group,mannitol group,high glucose group,small interfering RNA(siRNA)negative control group without target gene(siRNA group),30 nmol/L siRNA down-regulated NDRG1 genome(siNDRG1 group)and 50 nmol/L siNDRG1 group.Normal group cells were cultured conventionally.The mannitol group was added with 25 mmol/L mannitol,and the high-glucose group was added with 25 mmol/L glucose.In the siRNA group,25 mmol/L glucose was added,and then blank siRNA was added for induction.The 30 and 50 nmol/L siNDRG1 groups were added with 25 mmol/L glucose and induced with 30 and 50 nmol/L siRNDRG1,respectively.All cells were incubated for 24 h for follow-up experiments.Cell proliferation was observed by 4',6-diaminidine 2-phenylindole staining.Cell counting kit-8 staining was used to detect cell activity.The expression level of NDRG1 mRNA and protein was detected by Western blot and realtime quantitative polymerase chain reaction.Cell migration was observed by cell scratch assay.Cell lumen formation assay was used to detect lumen formation.The two-tailed Student t test was used to compare the two groups.One-way analysis of variance was used to compare groups.Results There were significant differences in cell proliferation rate(t=36.659,57.645)mobility rate(t=24.745,33.638)and lumen formation number(t=41.276,22.867)between high glucose group and normal group and mannitol group(P<0.01).Compared with normal group and mannitol group,the relative expression levels of NDRG1gene mRNA and protein in high glucose group were significantly decreased,with statistical significance(t=46.145,21.541,36.738,32.976;P<0.001).Compared with the siRNA negative group,the relative expression levels of NDRG1gene mRNA and protein in 30 nmol/L siNDRG1 group and 50 nmol/L siNDRG1 group were significantly decreased,and the differences were statistica

关 键 词：NDRG1 视网膜血管内皮细胞 细胞增殖 细胞迁移 细胞管腔形成 新生血管 细胞实验 

分 类 号：R774.1[医药卫生—眼科]