DNP辐射增敏及其相关机制研究
发布时间：2018-07-14 22:28
【摘要】：背景及研究目的:DNP(2,4二硝基苯酚)是一种能抑制氧化磷酸化的解偶联剂,能通过抑制ADP的磷酸化过程而抑制细胞的能量代谢,而只能以热能的方式散发出去,曾经作为减肥药物而广泛使用。肿瘤细胞是一种高耗能的细胞,同时肿瘤细胞受到射线照射以后,其自身的生长增殖以及对辐射损伤的修复都需要大量的能量。因此可以设想使用DNP,通过抑制细胞能量的生成,抑制受损伤肿瘤细胞的修复增殖,从而达到辐射增敏的效果。本文拟通过研究DNP对Hela和KB细胞的效应,评价该药物的辐射增敏能力,探讨其产生增敏效应的可能相关机制,为研发新的辐射增敏药物提供实验数据。方法:MTT实验和细胞克隆形成实验检测不同浓度的DNP对Hela以及KB的生长和增殖能力的影响。然后选择200μmol/L DNP浓度继续实验,通过细胞克隆形成实验,证实DNP是否能够诱导辐射增敏效应;通过流式细胞仪,检测对Hela、KB细胞的周期分布影响和线粒体膜电位的改变;通过使用PDMPO溶酶体黄/蓝色荧光探针,激光共聚焦显微镜观察细胞溶酶体内p H变化;通过透射电子显微镜观察药物作用后Hela和KB细胞的亚细胞结构的变化,尤其是自噬小体和自噬溶酶体堆积的形成。结果:DNP对Hela和KB细胞生长增殖具有明显的抑制作用,KB细胞更为敏感;DNP具有明显的辐射增敏作用,对两种细胞的增敏比SERD0分别为1.45和1.18。Hela和KB细胞在单纯药物作用24h后,或者联合4 Gy X线照射后,细胞G2/M期的细胞比例明显上升,产生了G2/M期的细胞阻滞。线粒体膜电位检测也发现,无论是单独药物作用,单纯4Gy照射,或者4Gy X线照射后联合药物作用24h,均能使低线粒体膜电位的细胞比例升高,药物联合射线后效果更加显著。与对照组相比,DNP作用后的Hela、KB细胞内均发现自噬小体和溶酶体聚集,KB细胞更加明显增多;KB细胞溶酶体内PDMPO溶酶体黄/蓝色荧光探针的蓝色荧光强度稍高于对照KB细胞组,说明药物作用后,溶酶体的p H值也受到了影响,而Hela细胞未能明显观察到荧光的显著变化。结论:DNP能够诱导产生辐射增敏效应,其作用机制可能包括:DNP诱导细胞G2/M期周期阻滞,导致细胞线粒体膜电位显著下降而抑制线粒体功能,细胞溶酶体p H向偏中性变化,增加细胞自噬效应,促进细胞启动死亡过程等。DNP可能是一种有前途的辐射增敏剂。
[Abstract]:Background and objective: DNP is a kind of uncoupling agent that can inhibit oxidative phosphorylation. It can inhibit the energy metabolism of cells by inhibiting the phosphorylation of ADP, but it can only be emitted in the form of heat energy. It was widely used as a weight loss drug. Tumor cell is a kind of high energy consuming cell. At the same time, the growth and proliferation of tumor cells and the repair of radiation damage require a lot of energy. It can be envisaged that DNPs can inhibit the repair and proliferation of damaged tumor cells by inhibiting the generation of cell energy, thus achieving the effect of radiosensitization. In this paper, the effects of DNP on Hela and KB cells were studied, and the radiosensitization ability of DNP was evaluated, and the possible mechanism of its sensitization effect was discussed, which could provide experimental data for the development of new radiosensitizing drugs. Methods the effects of different concentrations of DNP on the growth and proliferation of Hela and KB were detected by cell clone formation and cell MTT assay. Then the concentration of 200 渭 mol / L DNP was selected to continue the experiment, the cell clone formation test was used to confirm whether DNP could induce radiosensitization, and the effect of DNP on the cell cycle distribution and mitochondrial membrane potential was detected by flow cytometry. By using PDMPO lysosomal yellow / blue fluorescence probe, laser confocal microscopy was used to observe the changes of pH in lysosomes, and transmission electron microscopy was used to observe the changes of subcellular structure of Hela and KB cells. In particular, the formation of autophagy bodies and autophagy lysosomes. Results the growth and proliferation of Hela and KB cells were significantly inhibited by: 1: DNP. The sensitizing ratio of DNP to the two cells was 1.45 and 1.18. Hela and KB cells were respectively 1.45 and 1.18. Hela and KB cells were exposed to drugs alone for 24 hours. Or combined with 4 Gy X ray irradiation, the proportion of cells in G 2 / M phase increased significantly, resulting in cell arrest in G 2 / M phase. It was also found that the ratio of cells with low mitochondrial membrane potential (MMP) could be increased by the combination of 4 Gy alone or 4 Gy X ray irradiation for 24 h, and the effect was more significant after the combination of drug and radiation. Compared with the control group, there were more PDMPO lysosomal yellow / blue fluorescence probes in the lysosomal PDMPO fluorescence probe of the KB cells than those in the control group, and the fluorescence intensity of PDMPO lysosomal yellow / blue fluorescent probe was slightly higher than that of the control KB cell group, and the number of lysosome and lysosomal aggregating KB cells in the control group was higher than that in the control group. The results showed that the pH value of lysosome was also affected after the drug treatment, but the fluorescence of Hela cells was not observed significantly. ConclusionDNP can induce radiosensitization, and its mechanism may include G 2 / M cycle arrest induced by w DNP, resulting in a significant decrease in mitochondrial membrane potential and inhibition of mitochondrial function, and the neutral change of lysosome pH in cells. DNP may be a promising radiosensitizer to increase autophagy and promote cell death.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R73-36
本文编号：2123160
[Abstract]:Background and objective: DNP is a kind of uncoupling agent that can inhibit oxidative phosphorylation. It can inhibit the energy metabolism of cells by inhibiting the phosphorylation of ADP, but it can only be emitted in the form of heat energy. It was widely used as a weight loss drug. Tumor cell is a kind of high energy consuming cell. At the same time, the growth and proliferation of tumor cells and the repair of radiation damage require a lot of energy. It can be envisaged that DNPs can inhibit the repair and proliferation of damaged tumor cells by inhibiting the generation of cell energy, thus achieving the effect of radiosensitization. In this paper, the effects of DNP on Hela and KB cells were studied, and the radiosensitization ability of DNP was evaluated, and the possible mechanism of its sensitization effect was discussed, which could provide experimental data for the development of new radiosensitizing drugs. Methods the effects of different concentrations of DNP on the growth and proliferation of Hela and KB were detected by cell clone formation and cell MTT assay. Then the concentration of 200 渭 mol / L DNP was selected to continue the experiment, the cell clone formation test was used to confirm whether DNP could induce radiosensitization, and the effect of DNP on the cell cycle distribution and mitochondrial membrane potential was detected by flow cytometry. By using PDMPO lysosomal yellow / blue fluorescence probe, laser confocal microscopy was used to observe the changes of pH in lysosomes, and transmission electron microscopy was used to observe the changes of subcellular structure of Hela and KB cells. In particular, the formation of autophagy bodies and autophagy lysosomes. Results the growth and proliferation of Hela and KB cells were significantly inhibited by: 1: DNP. The sensitizing ratio of DNP to the two cells was 1.45 and 1.18. Hela and KB cells were respectively 1.45 and 1.18. Hela and KB cells were exposed to drugs alone for 24 hours. Or combined with 4 Gy X ray irradiation, the proportion of cells in G 2 / M phase increased significantly, resulting in cell arrest in G 2 / M phase. It was also found that the ratio of cells with low mitochondrial membrane potential (MMP) could be increased by the combination of 4 Gy alone or 4 Gy X ray irradiation for 24 h, and the effect was more significant after the combination of drug and radiation. Compared with the control group, there were more PDMPO lysosomal yellow / blue fluorescence probes in the lysosomal PDMPO fluorescence probe of the KB cells than those in the control group, and the fluorescence intensity of PDMPO lysosomal yellow / blue fluorescent probe was slightly higher than that of the control KB cell group, and the number of lysosome and lysosomal aggregating KB cells in the control group was higher than that in the control group. The results showed that the pH value of lysosome was also affected after the drug treatment, but the fluorescence of Hela cells was not observed significantly. ConclusionDNP can induce radiosensitization, and its mechanism may include G 2 / M cycle arrest induced by w DNP, resulting in a significant decrease in mitochondrial membrane potential and inhibition of mitochondrial function, and the neutral change of lysosome pH in cells. DNP may be a promising radiosensitizer to increase autophagy and promote cell death.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R73-36
【相似文献】
相关期刊论文 前4条
1 杨光伟,高时荣,冯俊光,程小曲,骆福添;临床放射治疗中增敏比的计算法[J];肿瘤研究与临床;2002年01期
2 金一尊,徐承熊;英国辐射增敏研究介绍[J];国外医学(放射医学分册);1986年04期
3 夏寿萱;日本辐射增敏研究概况[J];国外医学(放射医学分册);1986年01期
4 ;[J];;年期
相关硕士学位论文 前1条
1 李晓俊;DNP辐射增敏及其相关机制研究[D];苏州大学;2016年
，本文编号：2123160
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2123160.html
