ERK1/2信号通路的活化参与人脐静脉内皮细胞向间充质转分化的过程

ERK1/2 Signaling Pathway Is Involved in Endothelial-to-mesenchymal Transition in the Human Umbilical Vein Endothelial Cells

目的建立人脐静脉内皮细胞(HUVECs)向间充质转分化的模型并探讨其可能的信号转导途径。方法以HUVECs为研究对象,分组如下:对照组、转化生长因子-β1(TGF-β1)模型组、TGF-β1+DMSO组以及TGF-β1+抑制剂干预组。TGF-β1模型组应用5ng/mL TGF-β1刺激HUVECs 72h;TGF-β1+DMSO组应用5ng/mL TGF-β1及1μL/mL DMSO刺激HUVECs 72h;TGF-β1+抑制剂干预组分别应用p38MAPK抑制剂SB203580(5mmol/L)或ERK抑制剂U0126(10mmol/L)或JNK抑制剂SP600125(5mmol/L)干预TGF-β1诱导的内皮细胞,倒置显微镜观察内皮细胞形态,应用免疫荧光法检测VE-钙粘蛋白(VE-cadherin)和α-肌动蛋白(α-SMA)在细胞内的分布情况;并应用Western blot法检测VE-cadherin和α-SMA的蛋白表达情况。结果 TGF-β1(5ng/mL)刺激HUVECs 72h后,内皮细胞形态由卵圆形铺路石状向梭形转变,与0h相比,VE-cadherin蛋白表达显著下调(P<0.05),α-SMA蛋白表达显著上调(P<0.05)。抑制ERK1/2信号转导通路,可维持HUVECs内皮细胞表型以及VE-cadherin在内皮细胞的表达,抑制α-SMA蛋白表达,与TGF-β1模型组相比,差异具有统计学意义(P<0.05);抑制p38MAPK和JNK通路,与TGF-β1模型组相比,HUVECs表型、VE-cadherin和α-SMA蛋白表达未见明显差异。结论 TGF-β1可诱导内皮细胞向间充质转分化,应用U0126抑制ERK1/2信号途径可抑制内皮细胞转分化的进展,提示ERK1/2的活化可能是该过程重要的信号转导途径。

Objective To establish a human umbilical vein endothelial cells （HUVECs） model of endothelial-to mesenchymal transition （EndoMT） and to explore the possible signal transduction pathways. Methods HUVECs were divided into four groups as follows ： control group;TGF-βl group,in which the cells were stimulated with 5 ng/mL TGF-β1 for 72 h;TGF-β1 plus DMSO group,in which the cells were treated with 5 ng/mL TGF-β1 and 1 μL/mL DMSO for 72 h;TGF-β1 plus MAPK inhibitor groups,in which the cells were treated with 5 ng/mL TGF 111 and MAPK specific inhibitors （p38 MAPK inhibitor,5 mmol/ L SB203580 ;JNK inhibitor,5 mmol/L SP600125;or ERK1/2 inhibitor, 10 mmol/L U0126）. The morphology of cells was observed under the inverted phase contrast microscope. The expression of VE-cadherin and a smooth muscle actin （α-SMA） was detected by Western blot and immunofluorescence staining. Results The morphology of HUVECs was transformed from cobblestone-like appearance to fibroblast-like one when they were treated with 5 ng/mL TGF-β1 for 72 h. The expression of VE-cadherin was significantly decreased and that of α-SMA significantly increased in TGF-β1 group compared with control group （P〈0.05）. When the ERK1/2 signaling pathway was inhibited, HUVECs maintained the morphology of endothelial cells and the expression of VE-cadherin,while the expression of a-SMA was downregulated significantly （P〈0.05）. There was a significant difference in the expression of VE-cadherin and α-SMA between TGF-β1 group and TGF-131 plus ERK1/2 inhibitor group （P〈0. 05）. No significant difference was found in the morphology of cells and the expression of VE cadherin and α-SMA between TGF-β1, group and TGF-131 plus p38 MAPK or JNK inhibitor group （P〉0.05）. Conclusion TGF-β1 can induce EndoMT in HUVECs and inhibiting ERK1/2 signaling pathway by U0126 can significantly attenuate the EndoMT, which suggest that ERK1/2 signaling pathway is involved in the EndoMT of HUVECs.