Smad核转录共抑因子在人近端小管上皮细胞转分化中的表达及作用

The Expression and Role of SnoN in Human Proximal Tubular Epithelial Cells Transition

目的:观察Smad核转录共抑因子(SnoN)在TGF-β1致人近端小管上皮细胞转分化过程中的表达,探讨SnoN蛋白对TGF-β1所致的小管上皮细胞转分化的作用。方法:体外培养的人近端小管上皮细胞(HK-2),随机分为正常对照组、TGF-β1(5ng/ml)组和TGF-β1(5ng/ml)+MG-132(5μmol/ml)组。Western-blot检测细胞中SnoN蛋白水平的改变和Smad2/3磷酸化水平的改变;半定量RT-PCR方法观察细胞中SnoN mRNA表达的改变。免疫荧光检测间充质细胞标记物α-SMA的表达。结果:Western-blot的结果显示:(1)TGF-β1作用于细胞,SnoN蛋白表达迅速减少,10min为对照组的38%,并呈时间依赖进行性减少,60min较0min降低89%(P<0.01),24h有所恢复但仍较0min明显降低(P<0.01);TGF-β1+MG-132组中SnoN蛋白表达与对照组差异无统计学意义,与TGF-β1组相比,SnoN蛋白水平明显上调。(2)半定量RT-PCR显示:TGF-β1作用于细胞后,与SnoN蛋白表达不同,SnoN mRNA迅速升高,60min其表达增高近1倍(与0min比较,P<0.01);TGF-β1+MG-132组中SnoNmRNA的表达与TGF-β1组差异无统计学意义。(3)TGF-β1诱导细胞10min即可引发Smad2/3磷酸化(与0min比较,P<0.01),随着作用时间的延长,细胞中磷酸化的Smad2/3蛋白表达水平逐渐升高,TGF-β1+MG-132组中,Smad2/3磷酸化蛋白的表达减少80%(P<0.01)。(4)TGF-β1作用于细胞24h后免疫荧光检测到重新表达的α-SMA,TGF-β1+MG-132组α-SMA的表达被抑制。结论:TGF-β1可诱导SnoNmRNA表达上调,但SnoN蛋白的表达遭遇泛素系统的降解显著减少,SnoN蛋白水平的下调可能是TGF-β1导致小管上皮细胞纤维化作用的必要条件。

Objective：To investigate and identify the changes and role of smad transcriptional co-repressor SnoN expression in human proximal tubular epithelial cells.Methods：Human kidney proxima tubular cells（HK2）were cultured in different conditions：control groups,TGF-β1（5 ng/ml）groups,TGF-β1（5 ng/ml）＋MG-132（5 μmol/ml）groups.Western-blot were performed to test changes of SnoN protein and Smad2/3 phosphorylation,semi-quantitive RT-PCR were performed to test diversify of SnoNmRNA.Immunofluorescence was used to detect the expression of α-SMA.Results：The level of SnoN protein decreasd progressively in tubular cells after TGF-β1 induced,SnoN protein expression loss 60% at 10 min and decreased 89% at 1hr（compared with 0 min,P〈0.01）.The expression of phospho-Smad2/3 decreased 80% in MG-132＋TGF-βgroups,compared with TGF-β groups（P〈0.01）.Semi-quantitative RT-PCR showed SnoNmRNA was up-regulated rapidly by TGF-β1 induced in contrast to expression of SnoNprotein.No differences between MG-132＋ TGF-β1 groups and TGF-β1 groups.Compared with TGF-β1 groups（P〈0.01）,Smad2/3 phosphorylation and significant decreasing of SnoN protein was hinded by addition of MG-132 in MG-132＋TGF-β groups.Conclusion：Difference expression of SnoNprotein and SnoNmRNA is showed after TGF-β1 stimulating cells in this study.Downregulation of SnoNprotein expression is essencial in human proximal tubular epithelial cells transition.