ZEB1在大鼠肝星状细胞激活及转分化中的作用
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摘要
目的:本实验中利用ELISA、MTT和Western Blotting等实验检测的方法,探究乙醛刺激肝星状细胞活化过程中TGF-β信号通路相关的机制,并通过刺激肝星状细胞活化的方法来观察TGF-β信号通路的功能及效应。首先,乙醛刺激肝星状细胞株并设置不同的刺激时间,可通过MTT法来检测活化肝星状细胞的增殖水平;通过ELISA法测定肝星状细胞内纤维粘连素FN以及胶原蛋白I含量,证明乙醛能有效得刺激肝星状细胞株的活化,并比较不同刺激时间的差异。接着采用SB-431542特异性阻断TGF-β信号通路结合乙醛刺激HSC细胞,Western blotting检测HSC内ZEB1、α-SMA、Smad3和p-Smad3-S425的表达,以探究TGF-β信号通路相关机制。方法:①将细胞株分组为:(1)正常培养12小时,(2)正常培养24小时,(3)正常培养36小时,(4)TGF-β阻断正常培养24小时,(5)TGF-β阻断乙醛24小时刺激培养,(6)乙醛12小时刺激培养,(7)乙醛24小时刺激培养,(8)乙醛36小时刺激培养。培养相应组的细胞到细胞融合度为70%至80%时,用胎牛血清含量为0.4%的DMEM培养基培养12小时即是对各组细胞进行了同步化处理,之后再用胎牛血清含量为10%的DMEM完全培养基进行培养,其中24小时TGF-β阻断正常培养组细胞培养前先用抑制剂(SB-431542最终浓度是10μmol/L)进行处理,24小时乙醛刺激TGF-β阻断组给予乙醛刺激(最终浓度是200μmol/L)前先用对应的抑制剂(SB-431542最终浓度是10μmol/L)预处理1小时,刺激满12小时补加一次乙醛刺激,刺激时间一到就可收取细胞。然后对细胞总蛋白进行提取,用G250法测出细胞中总蛋白的含量,SDS-PAGE电泳对蛋白进行分离以及用Western Blotting实验方法检测出效应蛋白的含量,Western Blotting检测的目标有:ZEB1、α-SMA、Smad3和p-Smad3-S425的表达。然后将图谱经Quantity One软件进行处理和分析,根据Western膜上的蛋白条带相应灰度值进行比较分析。②HSC-T6细胞株分组仍然为:(1)正常培养12h,(2)正常培养24h,(3)正常培养36h,(4)TGF-β阻断正常培养24h,(5)TGF-β阻断乙醛24小时刺激培养,(6)乙醛12小时刺激培养,(7)乙醛24小时刺激培养,(8)乙醛36小时刺激培养。将HSC-T6细胞株接种到96孔培养板中,每组设有3复孔,培养各组细胞到细胞融合度为70%至80%时,用胎牛血清含量为0.4%的DMEM培养基培养12小时即是对各组细胞进行了同步化处理,之后再用胎牛血清含量为10%的DMEM完全培养基进行培养,其中24小时TGF-β阻断正常培养组细胞培养前先用相应抑制剂(SB-431542最终浓度是10μmol/L)进行处理,24小时乙醛刺激TGF-β阻断组给予乙醛刺激(最终浓度是200μmol/L)前先用相应的抑制剂(SB-431542最终浓度是10μmol/L)预处理1小时,然后每隔12小时补加一次乙醛刺激。各组细胞处理完毕后,细胞生长的情况即可通过光镜进行观察,并可通过ELLSA法检测胶原蛋白Ⅰ和纤维粘连素FN的含量变化,以及利用MTT法检测细胞活性有无变化。结果:①肝星状细胞经乙醛刺激后可被激活,激活表现为:胶原蛋白Ⅰ和纤维粘连素FN在ELLSA法检测中含量均有一定的增加,细
胞增值活性用MTT法检测得出增强很多。说明酒精的代谢产物乙醛可刺激肝星状细胞的活化。加入阻断剂后,MTT测定结果显示细胞活性明显下调,Ⅰ型胶原蛋白(TIMPⅠ)和纤维粘连素(FN)含量也明显下调。②正常培养对照组HSC中α-SMA、ZEB1和p-Smad3-S425的表达均有上调,说明HSC具有基础活性;乙醛刺激组较对照组显著上调α-SMA、ZEB1和p-Smad3-S425的表达;TGF-β通路阻断对α-SMA、ZEB1和p-Smad3-S425的表达上调均有明显的抑制作用。③刺激时间比较得出:各项指标的改变随刺激时间延长逐渐增高,至24h达到最高峰,36h略有下降。结论:①乙醛对肝星状细胞的活化有明显的刺激作用,乙醛激活HSC细胞后,可以使HSC细胞大量增殖并转分化。②乙醛激活肝星状细胞的增殖及转分化效应在24小时左右达最高峰。③乙醛可通过刺激TGF-β信号通路导致肝星状细胞激活并转分化,SB-431542特异性阻断TGF-β信号通路后可有效抑制乙醛激活HSC细胞。④TGF-β信号通路的激活,以及效应蛋白α-SMA和转录因子ZEB1表达上调,提示TGF-β信号通路与EMT存在相关性。
In this study, we use ELLSA, MTT and Western blotting to show mechanisms of TGF-βsignaling pathway in the activated hepatic stellate cells stimulated by acetaldehyde. The functions of the TGF-βpathways effects are also observed during hepatic stellate cell activation. Firstly, acetaldehyde is used to stimulate HSC cell for different time. MTT is applied to assay the proliferation level of activated HSC; ELISA is applied to detect the concentrations of fibronectin (FN) and TIMPⅠ. To confirm that acetaldehyde can stimulate the activation of hepatic stellate cells, SB-431542 is used to block TGF-βsignal pathway specifically, and Western blotting is applied to detect the expression of ZEB1,α-SMA, Smad3 and p-Smad3-S425 in the HSC, to show mechanisms of TGF-βsignal pathway. Methods:①Cells were divided into: (1) A control group cultured for 12h, (2) A control group cultured for 24h, (3) A control group cultured for 36h, (4) An experimental group with blocked TGF- beta signal pathway for 24h, (5) An experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h, (6) An experimental group with acetaldehyde stimulation for 12h, (7) An experimental group with acetaldehyde stimulation for 24h, (8) An experimental group with acetaldehyde stimulation for 36h. After the cells were cultured to 70%-80% confluences in 0.4% FBS DMEM medium to synchronize cells for 12h, the medium was replaced to 10% FBS DMEM complete medium. The inhibitors (SB-431542 final concentration is 10μmol / L) of the corresponding training were used to treat the experimental group with blocked TGF- beta signal pathway for 24h, experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h use the corresponding inhibitor (SB-431542 final concentration is 10μmol / L) pretreatment for 1h before the aldehyde stimulation (final concentration is 200μmol / L), time of additional stimulation is 12h,collect cells after the different stimulate times. Then the total cellular protein was extract, the protein content was detect by using G250, the total protein was separated by SDS-PAGE electrophoresis and the protein content was detected by Western blotting. to test targets including: The expression ofα-SMA, ZEB1,Smad3 and p-Smad3-S425 in the HSC. According to western blotting protein band intensity, Quantity One analysis software was used for processing.②HSC-T6 cells were also divided into: (1) A control group cutured for 12h, (2) A control group cutured for 24h, (3) A control group cutured for 36h, (4) An experimental group with blocked TGF- beta signal pathway for 24h, (5) An experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h, (6) An experimental group with acetaldehyde stimulation for 12h, (7) An experimental group with acetaldehyde
stimulation for 24h, (8) An experimental group with acetaldehyde stimulation for 36h. Inoculate HSC-T6 cells beads in 96-well plates, each group with 3 holes. After the cells were cultured to 70%-80% confluences in 0.4% FBS DMEM medium to synchronize cells for 12h, the medium was replaced to 10% FBS DMEM complete medium. The inhibitors (SB-431542 final concentration is 10μmol / L) of the corresponding training were used to treat the experimental group with blocked TGF- beta signal pathway for 24h, experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h use the corresponding inhibitor (SB-431542 final concentration is 10μmol / L) pretreatment for 1h before the aldehyde stimulation (final concentration is 200μmol / L), time of additional stimulation is 12h. Then observe the growth conditions of cells under the light microscope, detect the activity of cells by MTT, using Elisa detect the content of collagenⅠand fibronectin FN. Results:①After being activated by acetaldehyde, the activation of HSC cells were significantly increased using MTT assay, collagenⅠand fibronectin FN content increase in different degrees using Elisa. It indicates the alcohol metabolite acetaldehyde stimulated significantly on the activation of hepatic stellate cells. After using the SB431542 blocked TGF-βpathway, the result of MTT showed the activation of HSC cells were significantly down-regulated, TIMPⅠand FN were also significantly down-regulated.②In the control groups, the
expression ofα-SMA, ZEB1 and p-Smad3-S425 in the HSC were slightly increased. It explains that the control groups have basic activity; Comparing with control groups, activated groups were significantly up-regulated the expression ofα-SMA, ZEB1 and p-Smad3-S425 in the HSC; The groups of TGF-βblocked restrain the expression ofα-SMA, ZEB1 and p-Smad3-S425 obviously.③Through comparing the different stimulate times of HSC obtain the result that the expression of 24h groups as the highest,as comparing with 24h, 36h groups slightly decrease.
Conclusion:①Acetaldehyde can activate HSC cells obviously, after the HSC cells is activated by acetaldehyde, HSC cells begin proliferation and transdifferentiation.②The effect of proliferation and transdifferentiation in HSC cells which have been activated by acetaldehyde can reach to the peak when the stimulating time is 24h.③Acetaldehyde can lead to activation of hepatic stellate cells and transdifferentiation by stimulating TGF-βsignaling pathway. Blocking TGF-βsignaling pathway by SB-431542 can restrain the acetaldehyde activate HSC cells effectively.④The activation of TGF-βsignaling pathway and the expression ofα-SMA and ZEB1 up-regulated significantly suggest that TGF-βis related to the EMT in human liver fibrosis.
胞增值活性用MTT法检测得出增强很多。说明酒精的代谢产物乙醛可刺激肝星状细胞的活化。加入阻断剂后,MTT测定结果显示细胞活性明显下调,Ⅰ型胶原蛋白(TIMPⅠ)和纤维粘连素(FN)含量也明显下调。②正常培养对照组HSC中α-SMA、ZEB1和p-Smad3-S425的表达均有上调,说明HSC具有基础活性;乙醛刺激组较对照组显著上调α-SMA、ZEB1和p-Smad3-S425的表达;TGF-β通路阻断对α-SMA、ZEB1和p-Smad3-S425的表达上调均有明显的抑制作用。③刺激时间比较得出:各项指标的改变随刺激时间延长逐渐增高,至24h达到最高峰,36h略有下降。结论:①乙醛对肝星状细胞的活化有明显的刺激作用,乙醛激活HSC细胞后,可以使HSC细胞大量增殖并转分化。②乙醛激活肝星状细胞的增殖及转分化效应在24小时左右达最高峰。③乙醛可通过刺激TGF-β信号通路导致肝星状细胞激活并转分化,SB-431542特异性阻断TGF-β信号通路后可有效抑制乙醛激活HSC细胞。④TGF-β信号通路的激活,以及效应蛋白α-SMA和转录因子ZEB1表达上调,提示TGF-β信号通路与EMT存在相关性。
In this study, we use ELLSA, MTT and Western blotting to show mechanisms of TGF-βsignaling pathway in the activated hepatic stellate cells stimulated by acetaldehyde. The functions of the TGF-βpathways effects are also observed during hepatic stellate cell activation. Firstly, acetaldehyde is used to stimulate HSC cell for different time. MTT is applied to assay the proliferation level of activated HSC; ELISA is applied to detect the concentrations of fibronectin (FN) and TIMPⅠ. To confirm that acetaldehyde can stimulate the activation of hepatic stellate cells, SB-431542 is used to block TGF-βsignal pathway specifically, and Western blotting is applied to detect the expression of ZEB1,α-SMA, Smad3 and p-Smad3-S425 in the HSC, to show mechanisms of TGF-βsignal pathway. Methods:①Cells were divided into: (1) A control group cultured for 12h, (2) A control group cultured for 24h, (3) A control group cultured for 36h, (4) An experimental group with blocked TGF- beta signal pathway for 24h, (5) An experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h, (6) An experimental group with acetaldehyde stimulation for 12h, (7) An experimental group with acetaldehyde stimulation for 24h, (8) An experimental group with acetaldehyde stimulation for 36h. After the cells were cultured to 70%-80% confluences in 0.4% FBS DMEM medium to synchronize cells for 12h, the medium was replaced to 10% FBS DMEM complete medium. The inhibitors (SB-431542 final concentration is 10μmol / L) of the corresponding training were used to treat the experimental group with blocked TGF- beta signal pathway for 24h, experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h use the corresponding inhibitor (SB-431542 final concentration is 10μmol / L) pretreatment for 1h before the aldehyde stimulation (final concentration is 200μmol / L), time of additional stimulation is 12h,collect cells after the different stimulate times. Then the total cellular protein was extract, the protein content was detect by using G250, the total protein was separated by SDS-PAGE electrophoresis and the protein content was detected by Western blotting. to test targets including: The expression ofα-SMA, ZEB1,Smad3 and p-Smad3-S425 in the HSC. According to western blotting protein band intensity, Quantity One analysis software was used for processing.②HSC-T6 cells were also divided into: (1) A control group cutured for 12h, (2) A control group cutured for 24h, (3) A control group cutured for 36h, (4) An experimental group with blocked TGF- beta signal pathway for 24h, (5) An experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h, (6) An experimental group with acetaldehyde stimulation for 12h, (7) An experimental group with acetaldehyde
stimulation for 24h, (8) An experimental group with acetaldehyde stimulation for 36h. Inoculate HSC-T6 cells beads in 96-well plates, each group with 3 holes. After the cells were cultured to 70%-80% confluences in 0.4% FBS DMEM medium to synchronize cells for 12h, the medium was replaced to 10% FBS DMEM complete medium. The inhibitors (SB-431542 final concentration is 10μmol / L) of the corresponding training were used to treat the experimental group with blocked TGF- beta signal pathway for 24h, experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h use the corresponding inhibitor (SB-431542 final concentration is 10μmol / L) pretreatment for 1h before the aldehyde stimulation (final concentration is 200μmol / L), time of additional stimulation is 12h. Then observe the growth conditions of cells under the light microscope, detect the activity of cells by MTT, using Elisa detect the content of collagenⅠand fibronectin FN. Results:①After being activated by acetaldehyde, the activation of HSC cells were significantly increased using MTT assay, collagenⅠand fibronectin FN content increase in different degrees using Elisa. It indicates the alcohol metabolite acetaldehyde stimulated significantly on the activation of hepatic stellate cells. After using the SB431542 blocked TGF-βpathway, the result of MTT showed the activation of HSC cells were significantly down-regulated, TIMPⅠand FN were also significantly down-regulated.②In the control groups, the
expression ofα-SMA, ZEB1 and p-Smad3-S425 in the HSC were slightly increased. It explains that the control groups have basic activity; Comparing with control groups, activated groups were significantly up-regulated the expression ofα-SMA, ZEB1 and p-Smad3-S425 in the HSC; The groups of TGF-βblocked restrain the expression ofα-SMA, ZEB1 and p-Smad3-S425 obviously.③Through comparing the different stimulate times of HSC obtain the result that the expression of 24h groups as the highest,as comparing with 24h, 36h groups slightly decrease.
Conclusion:①Acetaldehyde can activate HSC cells obviously, after the HSC cells is activated by acetaldehyde, HSC cells begin proliferation and transdifferentiation.②The effect of proliferation and transdifferentiation in HSC cells which have been activated by acetaldehyde can reach to the peak when the stimulating time is 24h.③Acetaldehyde can lead to activation of hepatic stellate cells and transdifferentiation by stimulating TGF-βsignaling pathway. Blocking TGF-βsignaling pathway by SB-431542 can restrain the acetaldehyde activate HSC cells effectively.④The activation of TGF-βsignaling pathway and the expression ofα-SMA and ZEB1 up-regulated significantly suggest that TGF-βis related to the EMT in human liver fibrosis.
引文
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[16] Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1 [J]. Nat Cell Biol, 2008, 10(5): 593-601
[17] Park SM, Gaur AB, Lengyel E, Peter ME. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2 [J]. Genes Dev, 2008, 22(7): 894-907
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[21] Miettinen PJ,Ebner R,Lopez AR,Derynck R.TGF-beta induced transdiferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors [J].J Cell Biol,1994.127(6 Pt2):2021-2036
[22] Ju W,Ogawa A,Heyer J,et a1.Deletion of Smad2 in mouse liver reveals novel functions in hepatocyte growth and differentiation [J].Mol Cell Biol,2006,26(2):654
[23] Phanish MK,Wahab NA,Colville-Nash P,et a1.The diferential role of Smad2 and Smad3 in the regulation of profibrotic TGF-β1 responses in human proximal-tubule epithelial cells [J].Biochem J,2006,393(Pt 2):601-607
[24] Han G,Lu SL,Li AG,et a1.Distinct mechanisins of TGF-betal-mediated epithelial to mesenchymal transition and metastasis during skin carcinogenesis [J].J Clin Invest,2005,115(7):1714-1723
[25] Zhu S,Wu H,Wu F,et al.MicroRNA-21 targets tumor suppressor genes in invasion and metastasis [J].Cell Res,2008,18(3):350-359
[26] Davis BN,Hilyard AC,Lagna G,Hata A.Smad proteins control DROSHA-mediated microRNA maturation [J].Nature,2008,454(7200):56-61
[27] Vasko V,Espinosa AV,Scouten W,et al.Gene expression and functional evidence of epithelial-to-mesenchymal transition in papillary thyroid carcinoma invasion [J].Proc Natl Acad Sci USA, 2007,104(8):2803-2808
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