肾小管上皮间充质转化细胞模型中WT1和Pax2重新表达的研究

目的:探讨肾小管上皮间充质转化(EMT)细胞模型中胚胎发育基因WT1和Pax2的重新表达及其规律,并研究上调表达外源性WT1对肾小管上皮细胞株(NRK52E)的影响。方法:采用10 ng/ml IL-1α刺激体外培养的NRK52E细胞,建立EMT细胞模型。采用脂质体转染技术,将pRc/CMV-D-WT1质粒瞬时转染NEK52E细胞。分别提取EMT细胞模型和质粒转染组不同时间点细胞的RNA和蛋白质,采用RT-PCR和Western blot检测NEK52E细胞WT1、Pax2、上皮细胞标志E-cadherin和间充质标志α-SMA的表达,研究基因表达的时效性变化规律,并观察细胞的形态。结果:成功建立EMT细胞模型,该模型中E-cadherin的表达显著减少,α-SMA的表达显著增多,细胞发生明显的成纤维化样改变。在EMT细胞模型中,出生后关闭的WT1和Pax2获得重新表达,且Pax2的表达早于α-SMA和WT1。在NEK52E细胞中上调表达WT1,细胞表达α-SMA,不表达Pax2,同时E-cadherin的表达显著减少,细胞出现成纤维化样改变。结论:Pax2和WT1基因是EMT中的关键基因,在EMT发生时呈序列启动,Pax2在EMT中处于WT1上游。胚胎发育基因WT1和Pax2的重新表达可能是EMT的重要机制。

Study on WT1 and Pax2 Re-expression in the Cell Model of Renal Tubular Epithelial to Mesenchymal Transition

Objective:To investigate the re-expression of the embryonic genes WT1 and Pax2 in the cell model of renal tubular epithelial to mesenchymal transition(EMT),and to explore the affect of up-regulation WT1 on NRK52E cells.Methods:10ng/mL IL-1α was applied on NRK52E cells in vitro to build EMT cell model.pRc/CMV-D-WT1 plasmid was transfected into NEK52E cells with Lipofectamine 2000.RNA and protein were extracted at different time points respectively,and WT1,Pax2,the epithelial marker E-cadherin,the mesenchymal marker α-SMA mRNA and protein were detected using RT-PCR and Western blot,and the morphology of cells was observed.Results:EMT cell model had been established successfully.WT1 and Pax2 gene which closed since birth re-expressed in the model of EMT,and Pax2 expression was earlier than that of α-SMA and WT1.While WT1 expression increasing in NRK52E cells,there had no evidence of expression of Pax2 except α-SMA,and E-cadherin expression decreased significantly with fibrosis changes.Conclusion:WT1 and Pax2 are the key genes in EMT,starting expression in sequence when EMT occurs.Pax2 may be at the upper stream of WT1.Embryonic genes WT1 and Pax2 re-expression may be the important mechanism of EMT.