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25mmol/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Studentt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.
Abstract
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 real-time 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 statistically significant(t=44.275,40.7577,57.167,25.877;P＜0.01).Compared with normal group and siRNA group,cell mobility in 30 nmol/LsiNDRG1 group was increased,and the difference was statistically significant(t=57.562,49.522;P＜0.01).Compared with normal group and siRNA group,the number of cell lumen formation in 30 nmol/LsiNDRG1 group was significantly increased in the same field of vision,and the difference was statistically significant(t=63.446,42.742;P＜0.01).Conclusion Down-regulation of NDRG1 gene can improve the activity,migration and lumen formation of RF/6A cells under hyperglycemia.
基金项目
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)
NETL
NSTL
万方数据