血管紧张素Ⅱ参与肾小管上皮细胞转分化的实验研究

Experimental study of angiotensin Ⅱ(AngⅡ ) involved in renal epithelial-mesenchymal transition

目的:本文通过观察血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)影响肾小管上皮细胞转分化(epithelial-mesenchymal transition,EMT)的作用,以及和转化生长因子-β1(transforming growth factor,TGF-β1)作用的关系,探讨AngⅡ参与肾小管间质纤维化的作用机制。方法:以人肾小管上皮细胞株(human kidney cell)HKC细胞为研究对象,采用蛋白印迹等方法,观察AngⅡ(10-9、10-8、10-7、10-6mol/L),及其与TGF-β1共同作用对该细胞表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、E-钙黏蛋白(E-cadherin)和纤维连接蛋白(fibronectin,FN)的影响。明胶酶谱法检测细胞培养上清液中基质金属蛋白酶-2和基质金属蛋白酶-9(MMP-2和MMP-9)的变化,Boyden小室检测HKC细胞的迁移能力。结果:①单独应用AngⅡ不能够造成HKC细胞E-cadherin表达的变化,也不能诱导α-SMA表达,但是却能上调FN的表达;②与TGF-β1共同作用时能够加强TGF-β1影响E-cadherin,α-SMA和FN表达的作用;③AngⅡ能够增加HKC生成MMP-2和MMP-9;④AngⅡ能够(10-7和10-6mol/L)增加HKC细胞迁移至Boyden小室膜下侧面的数目。结论:①AngⅡ可以参与EMT过程,但不是导致EMT的关键因素;②AngⅡ能够以协同的方式参与TGF-β1导致的EMT,可能以此方式加重肾小管间质的纤维化。

Objective：To investigate the effect of Angiotensin Ⅱ （Ang Ⅱ ）on renal tubular Epithelial-Mesenchymal transition,evaluate the relationship between Ang Ⅱ and transforming growth factor-β1 （TGF-β1） and discuss the mechanism of Ang Ⅱ involved in tubulointerstitial fibrosis. Methods：Human proximal tubular epithelial HKC cells were taken as research objects. They was maintained in DMEM/F12 medium supplemented with 10% newborn calf serum, α-SMA,E-cadherin and FN triggered by Ang Ⅱ （10^-9, 10^-8, 10^-7,10^-6 mol/L）,and the combination of TGF-β1 were tested by Western blot. The changes of MMP-2 and MMP-9 in supernatant were detected by gelatin zymogramphy. The migration of HKC was assayed by Boyden chamber. Results： （1）In HKC induced by Ang Ⅱ only, the expression of α-SMA and E-cadherin proteins has no change, while FN was highly expressed. （2）α-SMA, E-cadherin and FN triggered by combination of TGF-β1 and Ang Ⅱ was up-regulated,and Ang Ⅱ could enhance the effect of TGF-β1. （3）Ang Ⅱ up-regulated MMP-2 and MMP-9 expressions of HKC. （4）Ang Ⅱ （ 10^-7, 10^-6 mol/L） could increase the number of cells that migrated across the filter and attached to the underside of boyden chamber. Conclusion： （1）Ang Ⅱ was involved in EMT,while it was not the critical factor of EMT. （2）Ang Ⅱ cooperated with TGF-β1 was involved in EMT and might aggravate tubulointerstitial fibrosis.