小干扰RNA对高糖诱导大鼠肾小管上皮细胞Snail 1表达的抑制作用

The Inhibitory Effects of Small Interference RNA to Snail 1 Expression in Rat Renal Tubular Epithelial Cells Induced by High Glucose

目的:观察小干扰RNA对高糖诱导大鼠肾小管上皮细胞Snail1表达的抑制作用及意义。方法:将原代培养肾小管上皮细胞分为5组,(1)对照组(含糖5.5 mmol/L);(2)高糖组(含糖25 mmol/L);(3)Snail1 siRNA处理组,转染Snail1 siRNA,6 h后更换为高糖(含糖25 mmol/L)培养;(4)Control siRNA处理组,转染Control siRNA作为阴性对照,6 h后换为高糖(含糖25 mmol/L)培养;(5)高渗组(含甘露醇19.5 mmol/L)。72 h后收集细胞,用Western blot检测Snail 1、Vimentin蛋白表达,用半定量RT-PCR检测Snail 1、Vimentin、成纤维细胞特异性蛋白-1(FSP-1)和纤维连接蛋白(FN)的mRNA表达。结果:肾小管上皮细胞转染Snail1 siRNA后,与高糖组比较Snail1mRNA和蛋白表达分别下降62%和68%(P<0.01);与高糖组比较,Snail1 siRNA处理组Vimentin、FSP-1、FN蛋白和(或)mRNA表达显著下调(P<0.01)。结论:(1)小干扰RNA能显著抑制高糖诱导大鼠肾小管上皮细胞Snail1表达;(2)Snail1参与了高糖作用下肾小管上皮细胞病理改变,并在细胞外基质的沉积中起重要作用。

Objective： To observe the inhibitory effect of small interference RNA（siRNA） to Snail 1 expression in rat renal tubular epithelial cells induced by high glucose and its significance.Methods： Primarily cultured renal tubular epithelial cells were incubated in serum-free DMEM for 24 hours,and then were divided into five groups and treated differently as follows：（1）Cells in control group（group C） were cultured in medium with glucose of 5.5 mmol/L（LGM）;（2）Cells in high glucose group（group H） cultured in medium with glucose of 25 mmol/L（HGM）;（3） Cells in Snail 1 siRNA treated group（group S） cultured in HGM in 6 hours after the transfection of Snail 1 siRNA;（4） Cells in non-specific siRNA treated group（group NS） cultured in HGM in 6 hours after the transfection of non-specific siRNA;（5） Cells in hyperosmosis group（group HS） cultured in medium with D-manntiol of 19.5 mMmol/L.All of the cells were harvested in 72 hours of incubation,and protein and/or mRNA levels of Snail 1,vimentin,fibroblast special protein-1（FSP-1） and fibronectin were determined with Western blot and semiquantitative RT-PCR.Results： Transfection caused decreases of Snai l 1 mRNA and its protein levels by 62% and 68%（P〈0.01）as compared with those in cells of group H;vimentin,FSP-1 and fibronectin mRNA in group S decreased by 59%,71% and 77%（P〈0.01） respectively when compared with those of group H;Conclusions：（1）Snail 1 siRNA is able to depress high-glucose-induced Snail 1 expression in tubular epithelial cells;（2）Snail 1 plays a critical role in the extracellular matrix deposition.