线粒体靶向香豆素的合成及抗肿瘤和辐射增敏作用的研究

发布时间：2018-07-04 09:33

本文选题：香豆素 + 辐射增敏　；参考：《北京协和医学院》2017年硕士论文

【摘要】：目的:香豆素类衍生物具有良好的抗肿瘤活性,但线粒体靶向香豆素类化合物未见报道。本文工作合成了携带不同靶向基团的线粒体靶向香豆素,并对其抗肿瘤作用和辐射增敏作用进行了研究。方法:通过将6-甲基香豆素溴化,随后将溴化后的产物与靶向基团连接得到线粒体靶向的香豆素。利用MTT法检测非靶向香豆素以及不同靶向基团的线粒体靶向香豆素对肿瘤细胞的抑制作用以及对正常细胞A31细胞的毒性。克隆形成实验考察非靶向香豆素以及不同靶向基团的线粒体靶向香豆素对细胞增殖的影响,采用多靶单击拟合模型方程计算(4-(二甲氨基)吡啶)甲基香豆素放射增敏比,评价增敏效果。DCFH-DA法检测非靶向香豆素以及不同靶向基团的线粒体靶向香豆素对肿瘤细胞ROS水平的影响。Rh123法检测非靶向香豆素以及不同靶向基团的线粒体靶向香豆素对肿瘤细胞线粒体膜电势的影响。Rh-2-AM法检测非靶向香豆素以及以三苯基膦为靶向基团的线粒体靶向香豆素对肿瘤细胞线粒体Ca2+浓度的影响。Hoechst 33258染色法测定非靶向香豆素以及不同靶向基团的线粒体靶向香豆素对肿瘤细胞凋亡的影响。NAO染色法测定非靶向香豆素以及以三苯基膦为靶向基团的线粒体靶向香豆素对肿瘤细胞线粒体质量的影响。结果:通过质谱,核磁共振氢谱和碳谱,确认了目标化合物结构。MTT结果显示,不同靶向基团的线粒体靶向香豆素能有效降低肿瘤细胞的存活率,在50-500μmol/L浓度范围内对正常细胞A31无明显毒性,而非靶向香豆素对肿瘤细胞存活率无明显影响。克隆形成实验表明不同靶向基团的线粒体靶向香豆素都可以有效抑制肿瘤细胞的增殖,而非靶向香豆素对肿瘤细胞增殖无明显影响。结果表明6-(4-(二甲氨基)吡啶)甲基香豆素化合物浓度为IC20时的辐射增敏比1.58,ROS实验结果表明,和照射组相比,6-(4-(二甲氨基)吡啶)甲基香豆素明显提高了 A549细胞中的ROS水平。而6-(烟酰胺)甲基香豆素和6-(三苯基膦)甲基香豆素,和对照组相比,则明显提高了肿瘤细胞内的ROS水平。而非靶向香豆素对肿瘤细胞内的ROS水平无明显影响。Rh123实验结果表明,与对照组相比,6-(烟酰胺)甲基香豆素和6-(三苯基膦)甲基香豆素有效地降低了肿瘤细胞的线粒体膜电势。而非靶向香豆素对肿瘤细胞线粒体膜电势无明显影响。线粒体Ca2+测定实验结果表明,6-(三苯基膦)甲基香豆素可以明显提高HeLa细胞中线粒体Ca2+浓度。而非靶向香豆素对HeLa细胞中线粒体Ca2+浓度无明显影响。Hoechst 33258染色结果表明,6-(烟酰胺)甲基香豆素和6-(三苯基膦)甲基香豆素可以诱导肿瘤细胞产生凋亡。非靶向香豆素不能有效诱导HeLa细胞凋亡。NAO染色实验结果表明,6-(三苯基膦)甲基香豆素有效地降低了HeLa细胞的线粒体质量。非靶向香豆素对HeLa细胞中线粒体质量无明显影响。结论:非靶向香豆素对HeLa细胞存活率,增殖,凋亡无明显抑制效果。6-(4-(二甲氨基)吡啶)甲基香豆素可以明显地提高A549细胞对放射线的敏感性。6-(烟酰胺)甲基香豆素通过提高ROS水平和降低线粒体膜电势,促使肿瘤细胞凋亡。6-(三苯基膦)甲基香豆素则可以通过提高肿瘤细胞中ROS水平、降低线粒体膜电势、提高线粒体Ca2+浓度和降低线粒体质量,促使肿瘤细胞凋亡。因此通过将非靶向香豆素设计成线粒体靶向药物可显著提高其抗肿瘤活性。
[Abstract]:Objective: coumarin derivatives have good antitumor activity, but mitochondria targeted coumarins have not been reported. In this paper, the mitochondrial targeting coumarin with different target groups was synthesized, and its anti-tumor effect and radiation sensitization were studied. Method: bromination of 6- methyl coumarin and then bromine The transformed product was linked with the target group to obtain the mitochondrial targeting coumarin. The inhibitory effect of the mitochondrial targeting coumarin on the tumor cells and the toxicity to the normal cell A31 cells were detected by the MTT method. The cloning and formation of the non targeting coumarin and the lines of different target groups were investigated. The effect of marinin on the proliferation of cells was measured by a multi target clicking model equation (4- (two methylamino) pyridine) methyl coumarin radiosensitization ratio, and the effect of.DCFH-DA method to detect the effect of non targeting coumarin and the mitochondrial target of different target groups on the ROS level of tumor cells by.Rh123 method was not detected. Effects of coumarin and different targeting groups targeting coumarin on the mitochondrial membrane potential of tumor cells.Rh-2-AM detection of non targeting coumarin and mitochondrial target of three phenyl phosphine as the target group on the concentration of mitochondrial Ca2+ in tumor cells by the.Hoechst 33258 staining method for the determination of non targeting coumarin The effect of coumarin on the apoptosis of tumor cells with different target groups,.NAO staining method was used to determine the effect of coumarin on the mitochondrial quality of tumor cells by targeting coumarin and the target group with three phenyl phosphine as the target group. Results: the target compound structure.MT was confirmed by mass spectrometry, nuclear magnetic resonance hydrogen spectrum and carbon spectrum. T results showed that the mitochondrial targeting coumarin of different target groups could effectively reduce the survival rate of tumor cells, and there was no obvious toxicity to normal cell A31 in the concentration range of 50-500 mu mol/L, but the non targeting coumarin had no significant effect on the survival rate of tumor cells. The proliferation of tumor cells was inhibited effectively, but the non targeting coumarin had no obvious effect on the proliferation of tumor cells. The results showed that the radiation sensitization ratio of 6- (4- (two methylamino) pyridine) methyl coumarin compound was 1.58. The results of ROS experiment showed that 6- (4- (two methylamino) pyridine) methyl coumarin increased A5 obviously compared with the irradiated group. The level of ROS in 49 cells. While 6- (nicotinamide) methylene coumarin and 6- (three phenyl phosphine) methyl coumarin, compared with the control group, significantly increased the level of ROS in the tumor cells. The non targeting coumarin did not significantly affect the ROS level in the tumor cells, and the.Rh123 experiment showed that the 6- (nicotinamide) methyl coumarin and 6- (nicotinamide) were compared with the control group. Three phenyl phosphine) methyl coumarin effectively reduced the mitochondrial membrane potential of tumor cells. Non targeting coumarin had no significant effect on the mitochondrial membrane potential of tumor cells. Mitochondrial Ca2+ assay showed that 6- (three phenyl phosphine) methylene coumarin could significantly increase the mitochondrial Ca2+ concentration in HeLa cells, but not targeted coumarin to HeLa The results of.Hoechst 33258 staining showed that 6- (nicotinamide) methylene coumarin and 6- (three phenyl phosphine) methyl coumarin could induce apoptosis in tumor cells. Non targeting coumarin could not effectively induce HeLa cell apoptosis.NAO staining experimental results showed that 6- (three phenyl phosphine) methyl coumarin was reduced effectively. The mitochondrial mass of HeLa cells was lower. The non targeting coumarin had no significant effect on the mitochondrial mass in HeLa cells. Conclusion: non targeting coumarin has no obvious inhibitory effect on the survival rate, proliferation and apoptosis of HeLa cells.6- (4- (two methylamino) pyridine) methyl coumarin can significantly increase the sensitivity of A549 cells to radiation sensitivity.6- (nicotinamide) methyl. Coumarin can increase ROS level and decrease mitochondrial membrane potential, and induce apoptosis of tumor cells.6- (three phenyl phosphine) methylene coumarin, which can improve the mitochondrial Ca2+ concentration and decrease the mitochondrial mass and induce apoptosis by improving the ROS level in tumor cells, so that the non targeting coumarin is designed. Mitochondrial targeting drugs can significantly improve its antitumor activity.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

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