三氧化二砷纳米粒对NB4细胞PTEN蛋白和线粒体凋亡途径的影响

发布时间：2018-06-02 09:31

  本文选题：三氧化二砷 + 纳米　； 参考：《吉林大学》2014年硕士论文

【摘要】：本实验采用溶胶-凝胶法制备As2O3纳米粒，通过MTT法比较As2O3纳米粒和As2O3对NB4细胞的增殖抑制作用；通过Hoechst和PI染色、流式细胞仪（Flow cytometry,FCM）观察检测As2O3纳米粒和As2O3对NB4细胞的凋亡作用；通过罗丹明123染色检测两种剂型对NB4细胞的线粒体膜电位的影响；通过Western blot观察两种剂型作用NB4细胞后细胞内p-PTEN、p-AKT、Bax、caspase-3、caspase-9和AIF的表达变化，初步探讨As2O3纳米粒对NB4细胞的增殖抑制和诱导凋亡的作用机制。 细胞增殖抑制实验结果显示As2O3纳米粒和As2O3对NB4细胞均可产生抑制作用，且呈时间浓度依赖性；药物浓度和作用时间相同时，As2O3纳米粒对NB4细胞的生长抑制作用明显强于As2O3。细胞凋亡实验结果显示，As2O3纳米粒和As2O3作用NB4细胞后主要表现为凋亡，相同作用时间下，随药物浓度增加，凋亡率增加，且纳米粒组凋亡率明显多于传统剂型As2O3组。罗丹明123染色实验结果显示两种剂型均可降低NB4细胞的线粒体膜电位，且呈浓度依赖性；相同药物浓度和作用时间下，，As2O3纳米粒对NB4细胞的作用明显强于As2O3。Western blot结果显示，与同浓度As2O3比较，1.5μmol/L、3μmol/L As2O3纳米粒能显著下调p-AKT和PTEN的表达；相同浓度时，As2O3纳米粒较As2O3明显增加p-PTEN和Bax的表达，且呈浓度依赖性；1.5μmol/L和3μmol/LAs2O3caspase-3、caspase-9的表达量呈浓度依赖性增加，1.5μmol/L As2O3纳米粒较同浓度三氧化二砷表达量增加，而3μmol/LAs2O3纳米粒组caspase-3、caspase-9的表达量较同浓度三氧化二砷降低。随后继续研究了AIF的表达变化，结果显示3μmol/L As2O3纳米粒组AIF的表达量增加。因此，根据实验结果推测三氧化二砷和低浓度三氧化二砷纳米粒主要通过依赖caspase的线粒体凋亡途径诱导NB4细胞凋亡，而高浓度三氧化二砷纳米粒主要通过非依赖caspase的线粒体凋亡途径诱导NB4细胞凋亡。 根据上述实验结果得出以下结论： ⑴As2O3纳米粒和As2O3对NB4细胞的生长有抑制作用，呈时间和浓度依赖性，且As2O3纳米粒对NB4细胞的作用明显强于As2O3； ⑵As2O3纳米粒和As2O3诱导NB4细胞凋亡呈浓度依赖性；且As2O3纳米粒的诱导凋亡作用更为显著； ⑶As2O3纳米粒对NB4细胞诱导凋亡作用强于As2O3，可能与上调p-PTEN的表达、抑制AKT的活化和启动线粒体凋亡途径有关。
[Abstract]:In this experiment, As2O3 nanoparticles were prepared by sol-gel method, and the inhibitory effects of As2O3 nanoparticles and As2O3 on the proliferation of NB4 cells were compared by MTT method, and the apoptotic effects of As2O3 nanoparticles and As2O3 on NB4 cells were observed by flow cytometry (FCM) and Hoechst and Pi staining. The effects of two dosage forms on mitochondrial membrane potential of NB4 cells were detected by rhodamine 123 staining, and the changes of expression of p-PTEN, p-AKTX, caspase-3, caspase-9 and AIF in NB4 cells were observed by Western blot. To explore the mechanism of As2O3 nanoparticles inhibiting proliferation and inducing apoptosis of NB4 cells. The results of cell proliferation inhibition test showed that both As2O3 nanoparticles and As2O3 could inhibit the growth of NB4 cells in a time and concentration dependent manner, and the inhibitory effect of As2O3 nanoparticles on the growth of NB4 cells was stronger than that of as 2O 3 at the same drug concentration and time. The results of apoptosis experiment showed that the apoptosis rate of NB4 cells was increased with the increase of drug concentration at the same time, and the apoptotic rate of As2O3 group was higher than that of As2O3 group. The results of Rhodamine 123 staining showed that the mitochondrial membrane potential of NB4 cells was decreased in a concentration-dependent manner, and the effect of as _ 2O _ 3 nanoparticles on NB4 cells was significantly stronger than that of As2O3.Western blot at the same concentration and time. Compared with the same concentration of As2O3, 1.5 渭 mol 路L ~ (-3) 渭 mol/L As2O3 nanoparticles could significantly down-regulate the expression of p-AKT and PTEN, and the same concentration of as _ 2O _ 3 nanoparticles could significantly increase the expression of p-PTEN and Bax in the same concentration of as _ 2O _ 3 nanoparticles compared with As2O3. In a dose-dependent manner, the expression of caspase-3 caspase-9 was increased in a dose-dependent manner by 1.5 渭 mol/L and 3 渭 mol / L as _ 2O _ 3 caspase-3 caspase-9 in a dose-dependent manner, and the expression of caspase-3 caspase-9 in 3 渭 mol/LAs2O3 nanoparticles was lower than that in the same concentration of arsenic trioxide. The expression of AIF was further studied. The results showed that the expression of AIF in 3 渭 mol/L As2O3 nanoparticles was increased. Therefore, according to the experimental results, it is inferred that arsenic trioxide nanoparticles and low concentration arsenic trioxide nanoparticles induce apoptosis of NB4 cells mainly through mitochondrial apoptosis pathway dependent on caspase. High concentration of arsenic trioxide nanoparticles mainly induce apoptosis of NB4 cells through mitochondrial apoptosis independent of caspase. Based on the above experimental results, the following conclusions are drawn: 1As2O3 nanoparticles and As2O3 inhibited the growth of NB4 cells in a time-and concentration-dependent manner, and the effect of As2O3 nanoparticles on NB4 cells was significantly stronger than that on as _ 2O _ 3 cells. The apoptosis of NB4 cells induced by 2As2O3 nanoparticles and As2O3 was concentration-dependent, and the apoptosis induced by As2O3 nanoparticles was more significant. The effect of 3As2O3 nanoparticles on apoptosis of NB4 cells is stronger than that of As2O3, which may be related to up-regulation of p-PTEN expression, inhibition of AKT activation and activation of mitochondrial apoptosis pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R96

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