摘要
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 TGF-β_1-induced epithelial to myofibroblast transition (EMT) of human kidney tubular cells (HKC) and its cellular smad mediated signaling pathway. Methodology: Using the epithelial marker E-cadherin expression and de novo expression-smooth muscle actin (SMA), the key marker of myofibroblast, as the parameters for EMT. The TGF-β_1 initiating smad signaling pathway was tested by mean of cell culture, western blot analysis and gene transfer etc. The plasmid of smad 7 expression (psmad 7) that contains full-length human smad 7 cDNA was transfered to HKC, to study the effect of overexpression of smad 7 on the action of TGF-β_1 on HKC. Results:①Time-course studies revealed that TGF-β_1 induces smad2/3 phosphorylation as early as 10mins and lasting 48hrs. ②TGF-β_1 down-regulated expression of E-cadherin of HKC at 6hrs and induced de novo expression of α-SMA after 24hrs in a dose-depended manner. ③ Over-expression of smad 7 in HKC that transferred with psmad 7 abrogated TGF-β_1 induced the expression of α-SMA and E-cadherin depression. ④However, either suppression of MAP kinase with specific chemical inhibitor (PD98059 or SC68376) or inhibition of PI3 kinase by Wortmannin obliterated the action of TGF-β_1 on HKC. Conclusion:These results suggest that TGF-β_1-induce renal tubular EMT is smad signaling pathway-depended manner. Blockage of smad signaling pathway by over-expression of smad 7 can obliterate TGF-β_1 induced EMT.
出处
《肾脏病与透析肾移植杂志》
CAS
CSCD
2005年第3期230-234,共5页
Chinese Journal of Nephrology,Dialysis & Transplantation
基金
国家自然科学基金项目(NO:30270616
30470800)
