促炎症消退介质D类消退素对肺癌细胞迁移及侵袭力的影响

发布时间：2017-12-31 16:17

本文关键词：促炎症消退介质D类消退素对肺癌细胞迁移及侵袭力的影响　出处：《扬州大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：观察促炎症消退介质D类消退素AT-RvD1对肺癌细胞A549转移能力的影响,同时探索核转录因子(Nrf2)及M2型丙酮酸激酶(Pkm2)在此过程中的作用,为更深入研究肺癌转移过程和炎症相关肿瘤的分子机制提供实验理论依据。方法：1、肿瘤的转移与上皮细胞间质转化(EMT)的关系极其密切,利用不同浓度转化生长因子TGF-β1构建EMT肺癌转移模型,分别利用荧光探针技术检测细胞内活性氧(ROS)水平,MTT法检测细胞增殖,划痕实验测迁移,Transwell法测侵袭,同时利用实时荧光定量PCR技术和免疫印迹技术对上皮细胞表型E-钙黏蛋白、间质细胞表型波形蛋白、Nrf2及Pkm2的表达进行检测。2、利用不同浓度AT-RvD1和哺乳动物雷帕霉素靶蛋白(mTOR)激活剂预刺激细胞30分钟,观察其对TGF-β1诱导的肺癌EMT过程的影响,分别比较细胞增殖性、迁移性,侵袭性的变化,检测细胞内ROS水平及相关基因蛋白的表达情况。结果：1、转化生长因子TGF-β1能明显下调E-钙黏蛋白和Nrf2的表达,上调波形蛋白和Pkm2的表达,增加细胞内ROS水平并呈浓度依赖,增强A549细胞的迁移及侵袭能力,但对细胞增殖无明显影响。2、D类消退素AT-RvD1能抑制TGF-β1诱导的EMT过程,上调E-钙黏蛋白和下调波形蛋白的表达,并且对A549细胞的迁移及侵袭能力有明显的抑制作用,但不影响细胞的增殖,研究结果提示,AT-RvD1的抑制作用可能与下调Nrf2和Pkm2的表达相关。3、哺乳动物雷帕霉素靶蛋白(mTOR)激活剂能逆转AT-RvD1抑制Pkm2的作用,并且下调E-钙黏蛋白的表达影响侵袭性,但尚未对细胞的增殖能力有明确影响。结论：1、D类消退素AT-RvD1能抑制TGF-β1诱导的肺癌EMT过程,抑制细胞的迁移及侵袭能力,但对细胞增殖无显著影响。2、AT-RvD1可能部分通过抑制mTOR信号进而下调Pkm2的表达来抑制TGF-β1诱导EMT过程,Nrf2表达降低可能与之相关。
[Abstract]:Objective: To observe the inflammatory resolution medium class D AT-RvD1 regression effect on metastasis of lung cancer cells A549, and explore the nuclear transcription factor (Nrf2) and type M2 pyruvate kinase (Pkm2) in this process, provide theoretical basis for further study on molecular mechanism of lung cancer metastasis related tumor and inflammation. Methods: 1, the metastasis of tumor and epithelial mesenchymal transition (EMT) extremely close relationship, transforming growth factor beta 1 TGF- to construct the EMT model of lung cancer metastasis using different concentrations of reactive oxygen, respectively using fluorescence probe technique in the detection of cell (ROS) level, MTT cell proliferation assay, scratch test migration, invasion test method Transwell at the same time, using real-time fluorescence quantitative PCR and Western blot on the epithelial cell phenotype of E- cadherin, interstitial cell phenotype of vimentin, expression of Nrf2 and Pkm2 were detected using different concentrations of.2, AT -RvD1 and mammalian target of rapamycin (mTOR) cell activator pre stimulation for 30 minutes, to observe the effects of TGF- beta 1 induced lung cancer EMT process were compared, cell proliferation, migration, changes of invasion, detection of intracellular ROS levels and relative gene expression. Results: 1, transformation the expression of growth factor beta 1 TGF- could downregulate E- cadherin and Nrf2, upregulation of the expression of vimentin and Pkm2, increase the level of ROS cells and in a concentration dependent enhancement, the migration and invasion of A549 cells, but had no obvious effect on cell proliferation of.2, D type of resolvin AT-RvD1 can inhibit TGF- beta 1 EMT by the upregulation of the expression of E- cadherin and downregulation of vimentin, and the migration and invasion ability of A549 cells was significantly inhibited, but does not affect cell proliferation, results suggest that the inhibition of AT-RvD1 might be associated with the down-regulation of Nrf Expression of.3 2 and Pkm2, the mammalian target of rapamycin (mTOR) activator AT-RvD1 can reverse the inhibitory effect of Pkm2, and the effects of down regulated expression of E- cadherin and aggressive, but there is a clear impact yet on cell proliferation. Conclusion: 1. D resolvin AT-RvD1 can inhibit TGF- beta 1 lung cancer EMT the process of induction, inhibition of migration and invasion of cells, but no significant effect of.2 on cell proliferation, AT-RvD1 inhibited TGF- induced EMT expression of beta 1 may part through inhibition of mTOR signaling and down-regulation of Pkm2, decreased Nrf2 expression may be associated with it.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R734.2

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