硫酸吲哚酚通过TGF-β1诱导腹膜间皮细胞转分化

Indoxyl sulfate induces transdifferentiation of peritoneal mesenchymal cells through TGF-β1

目的探讨转化生长因子β1(TGF-β1)在硫酸吲哚酚诱导腹膜间皮细胞转分化中的作用。方法对人腹膜间皮细胞株HMrSV5进行体外培养,先用含10%胎牛血清的RPMI1640培养液培养,分别在培养液中加入1000μmol/L的硫酸吲哚酚,4.25%腹膜透析液(PDF),2μmol/L TGF-β1受体抑制剂LY364947(TRI)+PDF,TRI+1000μmol/L的硫酸吲哚酚共培养。用免疫荧光法观察细胞在0、4、24、48、72 h的形态改变,用qRT-PCR方法测得各组各时间点TGF-β1、Twist1和E-钙粘蛋白(E-cadherin)基因相对表达量。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果硫酸吲哚酚剌激细胞后,细胞由铺路石样变细变长,呈长梭形改变,而TRI+1000μmol/L硫酸吲哚酚干预的细胞变化不明显。免疫荧光染色结果显示,硫酸吲哚酚干预细胞后,从24 h开始α-平滑肌肌动蛋白(α-SMA)的表达量增加,48 h表达最明显,而同时段硫酸吲哚酚+TRI干预细胞后,α-SMA表达相对减少。qRT-PCR结果显示,与对照比较,加入硫酸吲哚酚或PDF干预48 h后TGF-β1、Twist基因相对表达量(1.03±0.01,1.07±0.01比0.28±0.01)升高,E-cadherin基因相对表达量(0.86±0.02,0.88±0.02比3.34±0.02)下降(P<0.05);与硫酸吲哚酚培养液比较,加入硫酸吲哚酚+TRI培养48 h后细胞TGF-β1、Twist1基因相对表达量(0.14±0.00比1.03±0.01)降低,E-cadherin基因相对表达量(1.69±0.03比0.86±0.02)升高,差异有统计学意义(P<0.05)。与PDF培养比较,加入PDF+TRI培养48 h后细胞中TGF-β1、Twist1基因相对表达量(0.14±0.00比1.07±0.01)降低,E-cadherin基因相对表达量(1.80±0.03比0.88±0.02)升高,差异有统计学意义(P<0.05)。结论硫酸吲哚酚可通过上调TGF-β1基因表达来促进腹膜间皮细胞EMT,Twist1参与其中。

Objective To investigate the role of transforming growth factor(TGF-β1)in the induction of indoxyl sulfate in the transdifferentiation of peritoneal mesenchymal cells.Methods Human peritoneal mesenchymal cell line HMrSV5 was cultured in vitro using RPMI1640 medium containing 10%fetal bovine serum.1000μmol/L indoxyl sulfate and 4.25%peritoneal dialysate(PDF)were added into the medium,respectively.2μmol/L TGF-β1 receptor inhibitor LY364947(TRI)+PDF,TRI+1000μmol/L indoxyl sulfate co-culture.The morphologic changes of cells at 0,4,24,48 and 72 h were observed by immunofluorescence method.The relative expression levels of TGF-β1,Twist1 and E-cadherin at each time point were measured by qRT-PCR method.One-way analysis of variance was used for comparison between multiple groups,and SNK-q test was used for pairwise comparison between different groups.Results After the stimulation of indoxyl sulfate,the cells became thin and long like paving stones,but the changes were not obvious after the intervention of TRI+1000μmol/L indoxyl sulfate.Immunofluorescence staining showed that the expression of alpha-smooth muscle actin(α-SMA)was increased from 24 h after indoxyl sulfate intervention,and the expression was most obvious at 48 h,while the expression ofα-SMA was decreased after indoxyl sulfate+TRI intervention at the same time.The results of qRT-PCR showed that compared with the control,the relative expression of TGF-β1 and Twist(1.03±0.01,1.07±0.01 vs 0.28±0.01)gene was increased after 48 h intervention with indoxyl sulfate or PDF.E-cadherin(0.86±0.02,0.88±0.02 vs 3.34±0.02)gene expression was decreased(P<0.05);Compared with indoxyl sulfate culture medium,the relative gene expression of TGF-β1 and Twist1(0.14±0.00 vs 1.03±0.01)was decreased after being added with indoxyl sulfate+TRI culture for 48 h.The relative expression level of E-cadherin(1.69±0.03 vs 0.86±0.02)was increased,and the difference was statistically significant(P<0.05).Compared with PDF culture,TGF-β1 and Twist148 h(0.14±0.00 vs 1.07±0.01)gene relative expression levels were decreased after adding PDF+TRI culture for 48 h.The relative expression level of E-cadherin(1.80±0.03 vs 0.88±0.02)was increased,and the difference was statistically significant(P<0.05).Conclusion Indoxyl sulfate can promote EMT through upregulation of TGF-β1 gene expression,and Twist1 was involved in the process induced by indoxyl sulfate in peritoneal mesenchymal cells.