LeftyA蛋白对转化生长因子β1所致肾小管上皮细胞纤维化的影响

The effects of LeftyA on renal epithelial fibrosis induced by transforming growth factor-β1

目的 观察LeftyA蛋白对转化生长因子β1（TGF-β1）诱导人肾小管上皮细胞（HK-2）纤维化的影响,并初步探讨其作用机制.方法 体外培养人肾小管上皮细胞（HK-2）细胞,通过LipofectamineTM2000介导基因转染及药物筛选构建LeftyA蛋白稳定表达HK-2细胞系;TGF-β1（10ng/ml）刺激此细胞系,于0、6、12、24、48h通过Western blot分别检测E-钙粘蛋白（E-cadherin）、α-平滑肌肌动蛋白（α-SMA）及p-Smad2/3的表达变化,通过Real-time PCR检测Ⅰ型胶原（ColⅠ）及结缔组织生长因子（CTGF）基因的表达变化.结果 不同浓度的TGF-β1刺激24 h后HK-2细胞内E-eadherin、α-SMA蛋白的变化均表现出明显的剂量效应,而当TGF-β1浓度达到10ng/ml时,α-SMA蛋白合成达到平台期（P＞0.01）,因此本实验中以10ng/ml的TGF-β1作为刺激剂量;TGF-β1（10 ng/ml）刺激HK-2细胞6 h可引起E-cadherin表达下降（P＜0.01）,刺激24 h后可诱导其合成表达α-SMA（P＜0.05）,同时细胞内ColⅠ基因转录增加（P＜0.01）;正常HK-2细胞内没有p-Smad2/3的表达,TGF-β1（10 ng/ml）刺激30min即可诱导其激活表达,并在1 h时迅速达到高峰,其后开始下降;高表达LeftyA蛋白能够减轻TGF-β1所致上皮细胞转分化（EMT）及纤维化的程度,降低p-Smad2/3的表达高峰,并抑制CTGF的表达（P＜0.01）.结论 LeftyA蛋白能够通过抑制TGF-β1所致Smad2/3磷酸化及CTGF表达发挥抗肾小管上皮细胞纤维化的作用.

Objective To investigate the effects of Lefty A on renal epithelial fibrosis induced by transforming growth factor-β1 （TGF-β1）. Methods Human proximal tubular epithelial cells （HK2 cells） were transfected with lipofectamineTM2000 vectors to express Lefty A. The Lefty transfected cells were treated with TGF-β1 （10 ng/ml） for 48 h. The expression of E-cadherin, a-SMA, and psmad2/3 was examined with Western blotting. The mRNA expressions of collagen type Ⅰ and connective tissue growth factor （CTGF） were examined with real-time PCR. Results The inhibition of Ecadherin protein expression and induction of a-SMA protein expression were assessed in the HK-2 cells after TGF-β1 treatment; and this effect was still detectable till the concentration of TGF-β1 decreased to 2. 5 ng/ml. The induction of a-SMA expression reached a plateau when the cells were treated with 10 ng/ml of TGF-β1. In the 48 hours after TGF-β1 treatment （10 ng/ml）, a time-dependent decrease in E-cadherin expression and increase in a-SMA expression were found in HK-2 cells. Smad2/3 signa ling pathway was activated 30 minutes after TGF-β1 treatment; the activation peaked 1 hour later, and decreased thereafter. Compared to the non-transfected cells, in the Lefty A transfected HK-2 cells,the CTGF expression and Smad2/3 signaling pathway activation significantly decreased, and the extent of epithelial mesenchymal transition （EMT） and renal epithelial fibrosis also decreased. Conclusions Lefty A may play an important role in inhibiting or reversing the renal fibrosis induced by TGF-β1.