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目的:研究转化生长因子β1(TGFβ1)诱导人肾小管上皮细胞(HKC)转分化作用与其细胞内信号传递途径的关系。方法:以细胞间粘连蛋白(Ecadherin)和α平滑肌肌动蛋白(αSMA)作为肾小管上皮细胞转分化的观察指标,借助细胞培养、蛋白印迹和基因转染等方法,检测TGFβ1引发的Smad信号途径的变化,和TGFβ1诱导的肾小管上皮细胞转分化作用。将含有Smad7基因表达质粒转染HKC细胞,观察高表达的Smad7蛋白对TGFβ1作用的影响。结果:实验结果表明①TGFβ1作用HKC细胞10min即可磷酸化HKC细胞内Smad2/3蛋白,而且此作用可持续48h以上;②TGFβ1作用HKC细胞6h即可下调Ecadherin蛋白的表达,24h后能诱导该细胞表达αSMA,其作用呈明显的剂量依赖关系;③转染Smad7表达质粒的HKC细胞,可拮抗TGFβ1诱导HKC细胞表达αSMA以及下调Ecadherin蛋白表达的作用;④应用特异性MAP激酶抑制剂(PD98059和SC68376)和PI3激酶抑制剂(Wortmannin)均不能拮抗TGFβ1的作用。结论:TGFβ1诱导的上皮细胞转分化作用是依赖其引发的Smad信号途径,阻断该信号传递途径则能够拮抗TGFβ的作用。
Objective: To investigate the relationship between transdifferentiation of human renal tubular epithelial cells (HKC) induced by transforming growth factor-β1 (TGFβ1) and its intracellular signal transduction pathway. Methods: Ecadherin and α SMA were used as indicators of renal tubular epithelial cell transdifferentiation. Smad signaling pathway induced by TGFβ1 was detected by cell culture, Western blot and gene transfection TGFβ1-induced renal tubular epithelial cell transdifferentiation. The Smad7 gene expression plasmid was transfected into HKC cells to observe the effect of high expression of Smad7 protein on TGFβ1. Results: ①TGFβ1 could phosphorylate Smad2 / 3 protein in HKC cells 10 minutes after treatment with TGFβ1, and this effect can be sustained for more than 48 hours; ② TGFβ1 can down-regulate the expression of Ecadherin protein in HKC cells for 6 hours and induce it after 24 hours αSMA in a dose-dependent manner; ③Transfection of HKC cells transfected with Smad7 plasmid could antagonize TGFβ1-induced αSMA expression and down-regulate the expression of Ecadherin protein in HKC cells; ④Applied specific MAP kinase inhibitors (PD98059 and SC68376) And PI3 kinase inhibitor (Wortmannin) can not antagonize the role of TGFβ1. CONCLUSION: TGFβ1-induced epithelial cell transdifferentiation is dependent on the Smad signaling pathway that it initiates. Blocking this signaling pathway can antagonize TGFβ.