Wnt阻滞剂Dickkopf-1对肾小管上皮细胞转分化的抑制作用

The inhibitory effect of Wnt antagonist Dickkopf-1(Dkk-1) on transdifferentiation of renal tubular epithelial cells

目的研究Wnt阻滞剂Dickkopf-1(Dkk-1)对转化生长因子β1(TGF-β1)诱导的肾小管上皮细胞-间充质转分化的影响。方法体外培养人近端肾小管上皮细胞(HKC),收集后分3组:对照组细胞以含10%胎牛血清的培养基常规培养,TGF-β1组在常规培养基中加入TGF-β1(终浓度20ng/ml),TGF-β1+Dkk-1组同时加入TGF-β1(终浓度20ng/ml)和Dkk-1(终浓度100ng/ml)。培养48h后在倒置相差显微镜下进行形态观察,分别采用RT-PCR和Western blotting检测Wnt4、β-catenin、E-cadherin、α-SMA mRNA及蛋白表达情况,细胞免疫荧光染色观察E-cadherin和α-SMA的表达。结果与对照组比较,TGF-β1组和TGF-β1+Dkk-1组Wnt4 mRNA和蛋白表达水平均显著增高(P<0.05),后两组间差异无统计学意义(P>0.05)。β-catenin mRNA表达在各组之间差异无统计学意义(P>0.05)。β-catenin蛋白在对照组呈低表达,TGF-β1组表达明显上调,TGF-β1+Dkk-1组较TGF-β1组表达下调(P<0.05),与对照组比较差异无统计学意义(P>0.05)。E-cadherin mRNA和蛋白在对照组呈高表达,TGF-β1组表达明显降低,TGF-β1+Dkk-1组较TGF-β1组表达显著增高(P<0.05),与对照组比较差异无统计学意义(P>0.05)。α-SMA mRNA和蛋白表达在对照组和TGF-β1+Dkk-1组的表达均较TGF-β1组明显降低(P<0.05)。细胞免疫荧光染色显示E-cadherin和α-SMA表达与RT-PCR和Western blotting检测结果一致。结论 Wnt阻滞剂Dickkopf-1能抑制TGF-β1诱导的肾小管上皮细胞转分化。

Objective To investigate the effects of the Wnt antagonist Dickkopf-1（Dkk-1） on epithelial mesenchymal transdifferentiation in human proximal tubular epithelial cells induced by transforming growth factor-β1（TGF-β1）.Methods Human proximal tubular epithelial cells（HKC） were cultured in vitro and divided into three groups as follows： control group,TGF-β1 group,and TGF-β1＋Dkk-1 group.The cells in the control group underwent routine culture with medium containing 10% fetal calf serum.For the TGF-β1 group,TGF-β1（final concentration 20ng/ml） was added into the routine culture medium.For TGF-β1＋Dkk-1 group,TGF-β1（final concentration 20ng/ml） and Dkk-1（final concentration 100ng/ml） were added at the same time.After cultured for 48h,we performed morphologic observation using an inverted contrast microscope.RT-PCR and Western blotting were adopted to detect the expressions of Wnt4,β-catenin,E-cadherin,and α-SMA mRNA.E-cadherin and α-SMA expressions were detected by cell immunofluorescence.Results Compared with control group,the mRNA expression of Wnt4 and the protein expression of Wnt4 were significantly increased in TGF-β1 and TGF-β1＋Dkk-1 groups（P0.05）.There was no significant difference between two groups（P0.05）.There was no obvious difference between each group in mRNA expression of β-catenin（P0.05）.The β-catenin protein exhibited low expression in control group,whereas the expression significantly increased in TGF-β1 group.The expression of β-catenin in TGF-β1＋Dkk-1 group was lower than that in TGF-β1 group（P0.05）,but there was no significant difference between TGF-β1＋Dkk-1 group and control group（P0.05）.The mRNA and protein expression of E-cadherin were high in control group,but were significantly decreased in TGF-β1 group.Their expressions in the TGF-β1＋Dkk-1 group were increased compared with that in TGF-β1 group（P0.05）,however,there was no significant difference compared with control group（P0.05）.The mRNA and protein expression of α-SMA were decreased in control and TGF-β1＋Dkk-1 groups,compared with TGF-β1 group（P0.05）.Immune fluorescent staining showed that the expression of E-cadherin and α-SMA were consistent with the results of RT-PCR and Western blotting.Conclusion Wnt antagonist Dickkopf-1（Dkk-1） can inhibit epithelial mesenchymal transdifferentiation induced by TGF-β1.