敲低金属蛋白酶组织抑制物1(TIMP-1)抑制TGF-β1诱导的MRC-5人胚肺成纤维细胞增殖和胶原蛋白合成

Knocking down TIMP-1 inhibits the proliferation and collagen synthesis of MRC-5 human embryonic lung fibroblasts induced by TGF-β1

目的探讨敲低金属蛋白酶组织抑制物1(TIMP-1)对转化生长因子β1(TGF-β1)诱导的MRC-5人胚肺成纤维细胞生长的影响。方法将MRC-5细胞分为空白组、 TGF-β1组、转染阴性对照小干涉RNA的TGF-β1组和转染TIMP-1小干涉RNA(siTIMP-1)的TGF-β1组。噻唑蓝(MTT)法检测细胞活力,流式细胞术检测细胞周期分布, ELISA检测上清液肿瘤坏死因子α(TNF-α)含量, Western blot法检测TIMP-1、α平滑肌肌动蛋白(α-SMA)、 1型胶原蛋白(Col1)和β联蛋白(β-catenin)的蛋白水平。结果转染siTIMP-1组的MRC-5细胞TIMP-1蛋白水平明显降低。与空白组相比, TGF-β1组细胞活力明显升高、 G0/G1期细胞百分比明显降低、 S期和G2/M期细胞百分比明显升高, TNF-α含量明显升高,α-SMA、 Col1和β-catenin的蛋白水平均明显升高。与TGF-β1组相比,敲低TIMP-1水平后,细胞活力明显降低、 G0/G1期细胞百分比明显升高、 S期和G2/M期细胞百分比明显降低, TNF-α含量明显降低,α-SMA、 Col1和β-catenin蛋白表达均明显降低。转染阴性对照小干涉RNA的细胞与TGF-β1组各项指标均无明显差异。结论敲低TIMP-1水平可抑制MRC-5细胞增殖活性、减少胶原蛋白合成及TNF-α释放,可能与抑制Wnt/β-catenin信号通路有关。

Objective To investigate the effects of TIMP-1 gene expression inhibition on the cell growth in human embryonic lung fibroblast MRC-5 induced by tumor necrosis factor-α(TNF-α).Methods MRC-5 cells were divided into blank group,TGF-β1 group,TGF-β1 group transfected with negative control small interfering RNA,and TGF-β1 group transfected with TIMP-1 small interfering RNA(siTIMP-1).The cell viability was measured by MTT assay.The cell cycle of MRC-5 was analyzed by MTT assay and flow cytometry.The content of TNF-αin the supernatant was detected by ELISA,and the protein expression of TIMP-1,α-SMA,collagen 1 andβ-catenin were determined by Western blot analysis.ResultsThe expression level of TIMP-1 in the siTIMP-1 group was significantly lower than that in the blank group.Compared with the blank group,the cell viability in the TGF-β1 group was significantly improved;the percentage of the cells in G0/G1 phase was significantly raised,the percentages of the cells in S phase and G2/M phase were significantly increased.The content of TNF-αwas significantly elevated,the protein expression ofα-SMA,collagen-I andβ-catenin were significantly enhanced.Compared with TGF-β1 group,after knock-down of TIMP-1 gene expression,the cell viability in siTIMP-1 combined with TGF-β1 group was significantly inhibited,the percentage of the cells in G0/G1 phase was significantly raised,the percentages of the cells in S phase and G2/M phase were significantly decreased.The content of TNF-αwas significantly reduced,and the protein expression ofα-SMA,collagen-I andα-catenin was significantly depressed.There was no significant difference between the cells transfected with small interfering RNA of negative control and those of TGF-β1 group.ConclusionKnock-down of TIMP-1 gene expression inhibits the cell viability,cell cycle,collagen synthesis and the release of inflammatory factor TNF-αin the MRC-5 cells.The mechanism is related to the inhibition of Wnt/β-catenin signaling pathway.