转化生长因子β1诱导A549细胞上皮-间充质转化的时间相关性
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摘要
目的:探讨转化生长因子β1(TGF-β1)诱导A549细胞发生上皮-间充质转化(EMT)的时间相关性。方法:将体外培养的A549细胞分为空白组(加入F-12培养基)、模型组(加入10ng/ml TGF-β1)后继续培养。采用MTT实验检测细胞的增殖能力,光学显微镜下观察细胞形态学改变,实时荧光定量PCR和Western blot法检测E-Cad、Vim、FN mRNA的蛋白表达水平。结果:模型组在48h、72h时与空白组比较,A549细胞显著增殖(均P<0.01)、细胞形态明显改变,72h时几乎所有细胞呈纺锤形;48h、72h时与空白组比较,模型组FN mRNA表达显著上调(P<0.05,P<0.01),E-Cad mRNA表达显著下调(P<0.01)。48h时与空白组比较,模型组FN蛋白表达上调(P<0.05),E-Cad蛋白表达显著下调(P<0.01)。在72h时与空白组比较,模型组FN、Vim蛋白表达明显上调(P<0.01),E-Cad mRNA表达显著下调(P<0.01)。结论:A549细胞发生EMT与TGF-β1刺激的时间长短有关。72h和48h为体外实验EMT发生的时间,72h为EMT的最佳时间点。
Objective:To investigate the time-dependent property of transforming growth factor-β1(TGF-β1)induced epithelial-mesenchymal transition(EMT)in A549 cells.Methods:A549 cells were cultured in vitro,and the cells were divided into two groups:blank group was added with F-12 medium,and model group was added additionally with 10 ng/ml TGF-β1.The cells were cultured continuously for 24 h,48h and 72 h.Firstly,MTT assay was used to detect the proliferative capacity of the cells,and then the morphology of them was observed under light microscope.The protein expressions of E-Cad,Vim and FN m RNA were detected by realtime fluorescent quantitative PCR and Western blot.Results:A549 cells were significantly proliferated at 48 h and 72 h in model group,the differences between two groups were statistically significant(both P<0.01).Compared with blank group,the morphology of the cells changed significantly at 72 h.The expression levels of FN mRNA were up-regulated at 48 h and 72 h in the model group than those in the blank group(P<0.05,P<0.01)and the expression levels of E-Cad mRNA were down-regulated significantly(at 48 h and 72 h,both P<0.01).Compared with the blank group,the expressions of FN and Vim were up-regulated and E-Cad fluorescence expression was down-regulated in the model group.At 48 h compared with the blank group,FN protein expression was up-regulated in the model group(P<0.05),and E-Cad protein expression was down-regulated(P<0.01).Compared with the blank group at 72 h,the expressions of FN and Vim proteins were significantly upregulated in the model group(P<0.01),and the expression of E-Cad mRNA was significantly down-regulated(P<0.01).Conclusion:The experiment confirmed that the 72 h model of A549 is the best time point for TGF-β1 to induce EMT,suggesting that there is a time difference in the occurrence of EMT in vitro.
Objective:To investigate the time-dependent property of transforming growth factor-β1(TGF-β1)induced epithelial-mesenchymal transition(EMT)in A549 cells.Methods:A549 cells were cultured in vitro,and the cells were divided into two groups:blank group was added with F-12 medium,and model group was added additionally with 10 ng/ml TGF-β1.The cells were cultured continuously for 24 h,48h and 72 h.Firstly,MTT assay was used to detect the proliferative capacity of the cells,and then the morphology of them was observed under light microscope.The protein expressions of E-Cad,Vim and FN m RNA were detected by realtime fluorescent quantitative PCR and Western blot.Results:A549 cells were significantly proliferated at 48 h and 72 h in model group,the differences between two groups were statistically significant(both P<0.01).Compared with blank group,the morphology of the cells changed significantly at 72 h.The expression levels of FN mRNA were up-regulated at 48 h and 72 h in the model group than those in the blank group(P<0.05,P<0.01)and the expression levels of E-Cad mRNA were down-regulated significantly(at 48 h and 72 h,both P<0.01).Compared with the blank group,the expressions of FN and Vim were up-regulated and E-Cad fluorescence expression was down-regulated in the model group.At 48 h compared with the blank group,FN protein expression was up-regulated in the model group(P<0.05),and E-Cad protein expression was down-regulated(P<0.01).Compared with the blank group at 72 h,the expressions of FN and Vim proteins were significantly upregulated in the model group(P<0.01),and the expression of E-Cad mRNA was significantly down-regulated(P<0.01).Conclusion:The experiment confirmed that the 72 h model of A549 is the best time point for TGF-β1 to induce EMT,suggesting that there is a time difference in the occurrence of EMT in vitro.
引文
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[12]李婷,孟莹.上皮间质转化及其在肺间质纤维化作用的研究进展[J].国际呼吸杂志,2013,33(18):1433-1436.
[2]XU Xue-feng,DAI Hua-ping.Type 2 epithelial mesenchymal transition in vivo:truth or pitfalls?[J].Chin Med J,2012,125(18):3312-3317.
[3]Wills BC,Borok Z.TGF-beta-induced EMT:mechanisms and implications for fibrotic lung disease[J].Am J Physiol Lung Cell Mol,2007,293:L525-L534.
[4]Roberts AB,Tian F,Byfield SD,et al.Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition,fibrosis,tumor suppression and metastasis[J].Cytokine Growth Factor Rev,2006,17(1-2):19-27.
[5]沈克平,王海永,胡兵.上皮间质转化分子机制研究进展[J].世界中西医结合杂志,2011,6(1):87-88.
[6]Felton VM,Borok Z,Willis BC.N-acetylcysteine inhibits alveolar epithelial-mesenchymal transition[J].Am J Physiol Lung Cell Mol Physiol,2009,297:L805-L812.
[7]刘慧颖,殷正丰.上皮间质转化与循环肿瘤细胞[J].肿瘤,2013,33(9):4807-4840.
[8]张馨怡,薛辛东.上皮-间质转化的研究进展[J].国际儿科学杂志,2014,41(4):401-403.
[9]刘忠涛,熊力.TGF-β介导的上皮间质转化[J].中国普通外科杂志,2013,22(2):211-212.
[10]Sánchez-TillóE,Liu Y,de Barrios O,et al.EMT-activating transcription factors in cancer:beyond EMT and tumor invasiveness[J].Cell Mol Life Sci,2012,69:3429-56.
[11]Thiery JP,Acloque H,Huang RYJ,et al.Epithelial-mesenchymal transitions in development and disease[J].Cell,2009,139:871-890.
[12]李婷,孟莹.上皮间质转化及其在肺间质纤维化作用的研究进展[J].国际呼吸杂志,2013,33(18):1433-1436.
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