TGF-β1 induces activation of HSC-T6 cells and epithelial-mesenchymal transition in rats

目的探讨转化生长因子-β1（TGF-β1）在大鼠肝星状细胞系（HSC-T6）活化及上皮间质转换（EMT）中的作用。方法体外培养HSC-T6，用MTT法筛选TGF-β1对HSC-T6作用的最佳浓度；用10μg／L TGF-β1处理HSC-T624h，相差倒置显微镜下观察细胞形态改变，免疫荧光染色法检测细胞骨架结构F-actin蛋白的表达，RT-qPCR法检测肌动蛋白α-SMA及代表上皮间质转换的神经黏附素（N-cadherin）、波形蛋白（vimentin）和上皮黏附素（E-cadherin）基因表达；用不同浓度（0、5和10μg/L）的TGF-β1处理HSC-T624h，Western blot检测α-SMA、N-cadherin、vimentin和E-cadherin蛋白表达。结果10μg/L TGF-β1干预HSC-T624h有最好的细胞存活率；TGF-β1刺激HSC-T6后，细胞拉伸，伪足增多呈星形，细胞间连接疏松，呈显著活化状态；F-actin聚集形成应力纤维丝，沿细胞长轴分布；实验组α-SMA mRNA及vimentin mRNA的表达量明显高于对照组（P〈0．05），而E-cadherin mRNA的表达量明显降低（P〈0．05）；在不同浓度的TGF-β1呈剂量依赖性致α-SMA及N-cadherin和vimentin的蛋白表达量增多，而E-cadherin的蛋白表达量减少。结论TGF-β1可诱导HSC-T6活化及上皮间质转换。Objective To observe the effect of TGF-β1 on activation and epithelial mesenchymal transition （EMT） in rat hepatic stellate cell-T6. Methods Adopt the MTT method to screen the optimum concentration of TGF-β1 in vitro HSC-T6 cultured. After the HSC-T6 stimulation by TGF-β1 of 10 μg/L for 24 hours, the morphology of the cells was observed under inverted phase contrast microscope, the expression of F-actin which on behalf of cotoskeletal structure was detected by immunofluorescence staining; the expression of α-SMA and N-cadherin, vimentin, E-eadherin was measured by RT-qPCR ; The changes of α-SMA, N-cadherin, vimentin and E-eadherin were assessed by Western blot after different concentrations （0,5 and 10 μg/L） of TGF-β1 interventing HSC-T6 for 24 h. Results The optimal cell survival rate was recorded when 10 μg/L TGF-β1 dealt with cells for 24 h. After HSC-T6 were treated with TGF-β1 ,cells stretched, pseudopodia increased and turn into stellate, ceils connections were looser, so that represented a significantly activated state. F-actin filaments gathered to form stress and distributed along the long axis of the cells ; The expression of α-SMA mRNA and vimentin mRNA in experimental group was significantly higher while E-cadherin mRNA was obviously lower than the control group （P〈 0. 05）. TGF-β1 made the protein expression of α-SMA and N-cadherin, vimentin in dose-dependent increased while E-cadherin was decreased.Conclusions TGF-β1 may induce activation and epithelial-mesenchymal transition of HSC-T6.福建省科技计划（2015Y01010224）;福州市科技计划（2014-S-137-1

TGF-beta1 induces activation of HSC-T6 cells and epithelial-mesenchymal transition in rats

目的探讨转化生长因子-beta1(TGF-beta1)在大鼠肝星状细胞系(HSC-T6)活化及上皮间质转换(EMT)中的作用。方法体外培养HSC; -T6,用MTT法筛选TGF-beta1对HSC-T6作用的最佳浓度;用10 mug/L TGF-beta1处理HSC-T6 24; h,相差倒置显微镜下观察细胞形态改变,免疫荧光染色法检测细胞骨架结构F-actin蛋白的表达,; RT-qPCR法检测肌动蛋白alpha-SMA及代表上皮间质转换的神经黏附素(N-cadherin)、波形蛋白(vimentin)和上皮黏附素(; E-cadherin)基因表达;用不同浓度(0、5和10 mug/L)的TGF-beta1处理HSC-T6 24 h,Western; blot检测alpha-SMA、N-cadherin、vimentin和E-cadherin蛋白表达。结果10 mug/L; TGF-beta1干预HSC-T6 24; h有最好的细胞存活率;TGF-beta1刺激HSC-T6后,细胞拉伸,伪足增多呈星形,细胞间连接疏松,呈显著活化状态;F-actin聚集形成应力; 纤维丝,沿细胞长轴分布;实验组alpha-SMA mRNA及vimentin; mRNA的表达量明显高于对照组(P<0.05),而E-cadherin; mRNA的表达量明显降低(P<0.05);在不同浓度的TGF-beta1呈剂量依赖性致alpha-SMA及N-cadherin和vimentin; 的蛋白表达量增多,而E-cadherin的蛋白表达量减少。结论TGF-beta1可诱导HSC-T6活化及上皮间质转换。Objective To observe the effect of TGF-beta1 on activation and; epithelial mesenchymal transition(EMT) in rat hepatic stellate cell-T6.; Methods Adopt the MTT method to screen the optimum concentration of; TGF-beta1 in vitro HSC-T6 cultured. After the HSC-T6 stimulation by; TGF-beta1 of 10 mug/L for 24 hours, the morphology of the cells was; observed under inverted phase contrast microscope, the expression of; F-actin which on behalf of cotoskeletal structure was detected by; immunofluorescence staining; the expression of alpha-SMA and; N-cadherin,vimentin,E-cadherin was measured by RT-qPCR; The changes of; alpha-SMA,N-cadherin,vimentin and E-cadherin were assessed by Western; blot after different concentrations (0,5 and 10 mug/L) of TGF-beta1; interventing HSC-T6 for 24 h. Results The optimal cell survival rate was; recorded when 10 mug/L TGF-beta1 dealt withcells for 24 h. After HSC-T6; were treated with TGF-beta1,cells stretched, pseudopodia increased and; turn into stellate, cells connections were looser, so that represented a; significantly activated state.F-actin filaments gathered to form stress; and distributed along the long axis of the cells; The expression of; alpha-SMA mRNA and vimentin mRNA in experimental group was significantly; higher while E-cadherin mRNA was obviously lower than the control; group(P<0.05). TGF-beta1 made the protein expression of alpha-SMA and; N-cadherin, vimentin in dose-dependent increased while E-cadherin was; decreased.Conclusions TGF-beta1 may induce activation and; epithelial-mesenchymal transition of HSC-T6.福建省科技计划; 福州市科技计