癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (6): 411-417.doi: 10.3969/j.issn.1004-616x.2017.06.002

• 论著 • 上一篇    下一篇

TGF-β1诱导上皮间质转化过程中线粒体合成与功能的改变

张佳欣, 刘颖, 张伟, 周庆彪, 孔德钦, 刘瑞, 海春旭   

  1. 空军军医大学军事预防医学系毒理学教研室, 陕西省自由基生物学与医学重点实验室, 陕西 西安 710032
  • 收稿日期:2017-05-15 修回日期:2017-09-11 出版日期:2017-11-30 发布日期:2017-11-30
  • 通讯作者: 刘瑞,E-mail:liurui123@fmmu.edu.cn;海春旭,E-mail:cx_hai@fmmu.edu.cn E-mail:liurui123@fmmu.edu.cn;cx_hai@fmmu.edu.cn
  • 作者简介:张佳欣,E-mail:466982095@qq.com。
  • 基金资助:
    国家自然科学基金(81573126);陕西省自然科学基金青年人才项目(2014JQ4141)

Alteration of mitochondrial synthesis and function in epithelial-mesenchymal transition by TGF-β1

ZHANG Jiaxin, LIU Ying, ZHANG Wei, ZHOU Qingbiao, KONG Deqin, LIU Rui, HAI Chunxu   

  1. Department of Toxicology, School of Public Health, Shaanxi Key Lab of Free Radical Biology and Medicine, Medical University of the Air Force, Xi'an 710032, Shaanxi, China
  • Received:2017-05-15 Revised:2017-09-11 Online:2017-11-30 Published:2017-11-30

摘要: 目的:研究在转化生长因子-β1(TGF-β1)诱导上皮间质转化过程中线粒体合成与功能的改变。方法:体外培养人肺癌A549细胞,经不同浓度(0、2.5、5、10、20、40 ng/mL) TGF-β1处理24和48 h后观察其形态变化;采用CCK-8法检测经TGF-β1处理48 h后A549细胞的活力;流式细胞术检测细胞凋亡、活性氧(ROS)、线粒体活性氧及线粒体膜电位的变化;酶标仪检测ATP含量;Western blot分别检测细胞中上皮间质转化(EMT)相关标记物(E-cadherin和α-SMA)及线粒体合成相关蛋白(NRF-1及mt-TFA)的表达水平。结果:与对照组相比,随着TGF-β1浓度增加,A549细胞活力逐步上升,呈剂量-效应关系(r=0.941,P < 0.05);5~20 ng/mL TGF-β1处理组凋亡率明显升高(P均 < 0.05);不同浓度TGF-β1处理A549细胞48 h后大部分细胞呈现明显的间质细胞形态,出现上皮间质转化;Western blot结果显示,与对照组比较,经TGF-β1处理48 h后上皮标记物E-cadherin蛋白表达水平逐渐降低(P < 0.05),间质标记物α-SMA表达水平逐渐升高(P < 0.05);流式细胞术检测结果显示,5~20 ng/mL TGF-β1处理后细胞内ROS含量上升(P < 0.05),线粒体活性氧上升(P < 0.05),线粒体膜电位荧光强度下降(P < 0.05),ATP含量降低(P < 0.05),线粒体合成相关蛋白NRF-1和mt-TFA表达水平均下调(P均 < 0.05)。结论:TGF-β1诱导细胞发生上皮间质转化过程中线粒体的合成与功能受到影响,并由此促进了EMT的发生。

关键词: 上皮间质转化, 活性氧, 线粒体, 转化生长因子-β1

Abstract: OBJECTIVE:To investigate changes of ROS and mitochondria on epithelial-mesenchymal transition. METHODS:Human lung cancer A549 cells were divided into control and treated groups. The latter were treated with different concentrations of TGF-β1 (0,2.5,5,10,20,40 ng/mL) and morphological changes were observed after 48 hours. In addition,cell viability was detected by using the CCK-8 kit and cell apoptosis by flow cytometry. DCFH fluorescent probe,MitoSOX fluorescent probe and Rhodamine-123 staining were used to detect ROS and changes in mitochondrial membrane potential, respectively. The content of ATP was detect by using the enhanced ATP kit. The epithelial-mesenchymal transition-related markers and mitochondrial-related proteins were detected by Western blot. RESULTS:The treated groups showed major differences from the control group:cell viability increased gradually and showed a dose-effect relationship with increased concentrations of TGF-β1 (r=0.941,P < 0.05);there was a significant difference on cell apoptosis after treatment with 5-20 ng/mL TGF-β1;mesenchymal cells were observed after treatment with TGF-β1 for 48 hours, indicating the appearance of epithelial mesenchymal transition;Western blot showed that the expression of epithelial marker E-cadherin decreased gradually and the expression of mesenchymal marker α-SMA increased gradually (P < 0.05);the detection of intracellular reactive oxygen showed that treatment with 5-20 ng/mL TGF-β1 induced generation of ROS and mitochondrial ROS (P < 0.05);mitochondrial membrane potential declined as indicated by fluorescence intensity (P < 0.05);the content of ATP also decreased (P < 0.05);and expression of mitochondrial-related proteins were downregulated (P < 0.05). CONCLUSION:TGF-β1 caused increase of reactive oxygen species which led to the occurrence of mitochondrial dysfunction and promotion of EMT.

Key words: epithelial mesenchymal transition, reactive oxygen species, mitochondrial dysfunction

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