二去水卫矛醇对肺癌NCI-H460细胞的影响及基于拓扑异构酶Ⅱ探讨其抗肿瘤作用

发布时间：2018-06-02 08:46

  本文选题：二去水卫矛醇 + 肺癌　； 参考：《广西医科大学》2017年硕士论文

【摘要】：目的:评价二去水卫矛醇(dianhydrogalactitol,DAG)对肺癌NCI-H460细胞的抗肿瘤作用,探讨其抗肿瘤作用机制。方法:(1)DAG对NCI-H460细胞增殖的影响。(1)采用CCK-8法、细胞克隆形成实验,计算增殖抑制率及半数抑制浓度IC50,评价DAG对NCI-H460细胞的增殖抑制作用。(2)采用显微拍照观察不同浓度DAG给药48 h后,肺癌NCI-H460细胞形态的改变。(2)DAG对NCI-H460细胞迁移的影响。(1)采用划痕实验评价DAG对NCI-H460细胞横向迁移的影响。(2)采用Transwell-migration小室模型评价DAG对NCI-H460细胞纵向迁移的影响。(3)DAG对NCI-H460细胞DNA、Ca~(2+)和线粒体膜电位的影响。(1)采用Hoechst 33342染色检测DAG对细胞核染色质的影响。(2)采用单细胞凝胶电泳检测DAG对细胞DNA的损伤作用。(3)采用Fluo-3 AM为Ca~(2+)荧光探针,检测DAG对细胞内Ca~(2+)的影响。(4)采用Rhodamine123(Rhm 123)为线粒体膜电位荧光探针,检测DAG对细胞线粒体膜电位的影响。(4)DAG对Topo Ⅱα、Topo Ⅱβ的影响。(1)采用Real-time PCR法检测拓扑异构酶Ⅱα(Topo Ⅱα)、拓扑异构酶Ⅱβ(Topo Ⅱβ)mRNA的表达水平。(2)采用Western blot法检测Topo Ⅱα、Topo Ⅱβ蛋白表达水平。(3)应用计算机模拟分子对接技术预测DAG与Topo Ⅱα、Topo Ⅱβ的相互作用。结果:(1)CCK-8法检测结果显示,DAG对NCI-H460细胞的体外抗肿瘤活性显著,与对照组比较有显著性差异,其48 h的IC50为9.68±1.02μg/mL。细胞克隆形成实验结果表明,DAG能持续抑制肿瘤细胞的增殖。(2)划痕实验和Transwell-migration小室检测结果表明,DAG能抑制肿瘤细胞的迁移。(3)Hoechst 33342检测细胞凋亡结果显示,DAG使细胞核染色质发生明显改变,与空白对照组比较,各给药组细胞均不同程度出现细胞变圆缩小、贴壁能力减弱、胞浆内颗粒边缘化甚至细胞破碎等损伤现象;此外,单细胞凝胶电泳检测出DAG可导致DNA损伤。Fluo-3 AM荧光染色结果显示,与空白对照组比较,各给药组荧光强度增强,即细胞内Ca~(2+)浓度增加,说明DAG诱导细胞凋亡与细胞内Ca~(2+)浓度增加有关;Rhm 123荧光染色结果显示,与空白对照组比较,各给药组荧光强度减弱,表明DAG使细胞线粒体膜电位下降,即DAG诱导细胞凋亡与线粒体膜电位下降有关。(4)Real-time PCR检测结果显示不同浓度的DAG均能明显降低Topo Ⅱα、Topo ⅡβmRNA表达量降低;Western blot检测表明,DAG明显使Topo Ⅱα、Topo Ⅱβ蛋白表达量降低,提示DAG诱导细胞凋亡与下调Topo Ⅱα、Topo Ⅱβ有关;计算机模拟分子对接显示DAG与Topo Ⅱα、Topo Ⅱβ有相互结合作用。结论:(1)DAG能显著抑制NCI-H460细胞的增殖。(2)DAG能显著抑制NCI-H460细胞的迁移。(3)DAG诱导细胞损伤与其改变细胞内Ca~(2+)浓度、线粒体膜电位及DNA损伤有关。(4)作用机制研究表明DAG能降低Topo Ⅱα、Topo ⅡβmRNA和蛋白水平,并与Topo Ⅱα、Topo Ⅱβ结合,最终可能导致DNA双链断裂,引起细胞死亡。
[Abstract]:Aim: to evaluate the antitumor effect of dianhydrogalactil DAG on NCI-H460 cells of lung cancer and its mechanism. Methods the effect of DAG on the proliferation of NCI-H460 cells was studied by CCK-8 assay. The cell clone formation assay was used to calculate the inhibitory rate of proliferation and IC50, and to evaluate the inhibitory effect of DAG on the proliferation of NCI-H460 cells. The effects of DAG on the proliferation of NCI-H460 cells were evaluated by microphotography for 48 h after administration of different concentrations of DAG. Morphological changes of NCI-H460 cells in lung cancer. Effect of DAG on the migration of NCI-H460 cells. (1) the effect of DAG on the lateral migration of NCI-H460 cells was evaluated by scratch assay. (2) Transwell-migration chamber model was used to evaluate the effect of DAG on the longitudinal migration of NCI-H460 cells. Hoechst 33342 staining was used to detect the effect of DAG on nuclear chromatin. The single cell gel electrophoresis (SCGE) was used to detect the damage of DAG to cell DNA. Fluo-3 AM was used as the Ca~(2) fluorescent probe. The effect of DAG on intracellular Ca~(2. (4) Rhodamine123(Rhm 123) was used as fluorescence probe of mitochondrial membrane potential. Detection of the effect of DAG on mitochondrial membrane potential. The effect of Topo 鈪，

本文编号：1968151