摘要目的 探讨Numb在大鼠肾小管上皮细胞转分化过程中的作用.方法 重组人转化生长因子β1(TGF-β1)刺激大鼠近端.肾小管上皮细胞(NRK52E细胞),不同浓度TGF-β1(0、1、5、10、15、20 μg/L)作用48 h与TGF-β1 10 μg/L作用不同时间(0、24、48、72 h)后,采用RT-PCR、Western印迹和免疫荧光染色分别检测NRK52E细胞内E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)和Numb的表达.采用RNA干扰技术下调Numb表达,Western印迹观察改变Numb水平对E-cadherin、α-SMA蛋白水平的影响.结果 TGF-β1以剂量及时间依赖的方式诱导NRK52E细胞E-cadherin蛋白表达下调,α-SMA蛋白表达增高.Numb蛋白的表达随TGF-β1浓度的增加而增高,在5、10、15和20 μg/L时分别为0 μg/L时的1.33倍(P=0.024)、1.39倍(P=0.035)、1.45倍(P=0.025)和1.51倍(P=0.000).而Numb蛋白和mRNA的表达亦随TGF-β1作用时间的延长而增高,作用24 h、48 h、72 h,Numb蛋白分别为0 h的1.48倍(P=0.046)、1.54倍(P=0.011)、1.79倍(P=0.028),Numb mRNA分别为0 h的1.56倍(P=0.012)、1.82倍(P=0.008)、1.82倍(P=0.002);同时Numb的分布也发生了改变,大量聚集在胞质中.下调Numb表达可以显著抑制TGF-β1诱导的α-SMA表达上调(为Numb表达正常时的18.1%,P=0.004)、E-cadherin表达下调(为Numb表达正常时的2.19倍,P=0.004).结论 Numb可以促进肾小管上皮细胞发生转分化.

abstractsObjective To explore the effect of Numb on tubular epithelial-to-mesenchymal transition (EMT) in rat proximal epithelial cells. Methods NRK52E cells were treated with different concentrations of recombinant human transforming growth factor-β1 (TGF-β1) (0, 1, 5, 10, 15, 20 μg/L) for 48 h or 10 μg/L TGF-β1 for different times (0, 24, 48, 72 h) in vitro. The expressions of E-cadherin, a-smooth muscle actin(α-SMA) and Numb in NRK 52E cells were detected by RT-PCR, Western blot and immunofluorescence staining. Meanwhile Numb siRNA oligo was transfected into NRK 52E cells with lipofectamine before TGF-β1 treatment, then Western blot was applied to detect the protein expression of E-cadherin, α-SMA and Numb in NRK52E cells. Results TGF-β1 could induce EMT in NRK52E cells in dose- and time-dependent manner. During the progress of TGF-β1-induced EMT, the protein expression of Numb in 5, 10, 15, 20 μg/L group was 1.33 folds (P=0.024), 1.39 folds (P=0.035), 1.45 folds (P=0.025), 1.51 folds (P=0.000) respectively as compared to 0 μg/L group. Likewise, the protein and mRNA expression of Numb in 24 h, 48 h, 72 h group was 1.48 folds (P=0.046) and 1.56 folds (P=0.012), 1.54 folds (P=0.011) and 1.82 folds (P=0.008), 1.79 folds (P=0.028) and 1.82 folds (P=0.002) respectively as compared to 0 h group. Moreover, large amount of Numb was accumulated in the cytoplasm. Down-regulation of Numb expression by siRNA transfection did not influence the basal expression of E-cadherin and α-SMA in NRK 52E cells, but attenuated the progression of EMT in NRK52E cells induced by TGF-β1. The up-regulation of α-SMA protein was reduced to 18.1% (P=0.004) while the down-regulation of E-cadherin protein was reversed to 2.19 folds (P=0.004). Conclusion Numb can promote EMT in rat proximal epithelial cells.