DNA甲基化转移酶1介导体外高糖刺激诱导的小鼠足细胞损伤的实验观察

Dnmt1mediates mice podocyte injury induced by high glucose in vitro

目的研究高糖刺激对体外培养的小鼠足细胞DNA甲基化转移酶(Dnmts)表达的影响及Dnmt1在高糖刺激诱导的小鼠足细胞损伤中的作用。方法以体外培养的小鼠永生化足细胞为研究对象,分为正常糖对照组、高糖刺激组、甘露醇组及高糖+干预组(DNA甲基化抑制剂5-氮杂-2′-脱氧胞苷,Dnmt1-siRNA),培养48h,RT-PCR检测其Dnmts mRNA表达;免疫印迹检测Dnmt1、nephrin及podocin蛋白表达;Transwell转板迁移实验观察不同处理后足细胞迁移数目。结果与正常糖对照组比较,高糖刺激组足细胞Dnmt1mRNA及蛋白表达增加(P<0.01),Dnmt3a及Dnmt3bmRNA表达未见明显改变。经5-氮杂-2′-脱氧胞苷或Dnmt1-siRNA处理48h后,高糖刺激组导致Dnmt1高表达得到抑制,降低的足细胞裂孔膜蛋白podocin、nephrin表达增加(P<0.01)。结论高糖上调足细胞Dnmt1蛋白表达,Dnmt1参与体外高糖诱导的小鼠足细胞损伤。

Objective To investigate the effect of high glucose stimulation on expression of Dnmts in cultured mice podocytes and the role of Dnmt1 in podocyte injury induced by high glucose. MethodsCultured mice glomerular podocyte cells were used and divided into four groups：control group coincubrated with normal glucose group,high glucose stimulation group,mannitol group and high glucose plus intervention group（5-aza-2′-deoxycytidine plus Dnmt1-siRNA）.All the groups were cultured for 48 h.Dnmts mRNA expression was detected by quantitative PCR.The protein levels of Dnmt1,nephrin and podocin were detected by western blot.Transwell migration assay were used to observe the number of podocyte migration. Results The mRNA and protein expression of Dnmt1 were increased in high glucose stimulation group than in normal glucose group,while the mRNA expression of Dnmt3 aand Dnmt3bwere not significantly changed.After 5-aza-2′-deoxycytidine or Dnmt1-siRNA treatment for 48 hours,high expression of Dnmt1 induced by high glucose was significantly inhibited,decreased slit diaphragm protein podocin and nephrin expression was significantly increased（P〈0.01）. ConclusionHigh glucose increases the mRNA and protein expression of Dnmt1.Dnmt1 is involved in podocyte injury induced by high glucose.