莫诺苷对SK-N-SH细胞氧化损伤的保护作用及机制研究

发布时间：2018-05-23 08:06

本文选题：莫诺苷 + SK-N-SH细胞　；参考：《蚌埠医学院》2015年硕士论文

【摘要】：目的:建立SK-N-SH细胞的过氧化氢(hydrogen peroxide,H2O2)的氧化损伤模型,初步探讨莫诺苷对细胞氧化损伤的保护作用。方法:(1)采用对数生长期的人神经母细胞瘤(SK-N-SH)细胞,给予不同浓度的莫诺苷(1μM,10μM和100μM)预处理24 h,加入H2O2(200-400μM)作用24 h以诱导细胞损伤。(2)在倒置相差显微镜下观察各组SK-N-SH细胞的生长和形态学变化。(3)用MTT法检测细胞存活率;乳酸脱氢酶(LDH)细胞毒性检测试剂盒检测细胞内LDH的释放量;脂质氧化(MDA)检测试剂盒检测细胞脂质氧化水平;超氧化物歧化酶(SOD)测定试剂盒检测细胞内SOD的含量。(4)以活性氧(ROS)检测试剂盒检测细胞内ROS的生成;超氧化物阴离子荧光探针(Dihydroethidium)检测细胞内超氧化物阴离子的水平。(5)应用线粒体膜电位检测试剂盒(JC-1)检测线粒体膜电位(△Ψm)的变化;采用分光光度法检测细胞内Caspase-3活性。(6)通过流式细胞术(FCM)、Hoechst染色分析细胞凋亡。(7)利用Western blot检测细胞凋亡相关蛋白的表达变化。(8)利用RT-PCR检测凋亡相关基因的表达。结果:1.MTT细胞活力检测结果显示,H2O2可降低SK-N-SH细胞活力,并呈现出剂量和时间依赖的关系。经过H2O2(200μM)处理24 h后,细胞活力较对照组显著降低,而莫诺苷能明显抑制H2O2导致的SK-N-SH细胞活力下降。2.形态学结果显示,经过H2O2(200μM)处理24 h后,SK-N-SH细胞可出现突起消失、胞体肿胀圆缩、部分细胞发生聚集并破裂成细胞碎片等显著的形态学变化;而莫诺苷的预处理能够减轻这一损伤变化。3.LDH细胞毒性检测结果表明,莫诺苷可减少胞内酶LDH的释放。4.流式细胞术和Hoechst染色的结果表明,莫诺苷可显著抑制H2O2诱导的SK-N-SH细胞凋亡。5.DCFH-DA和Dihydroethidium荧光探针检测结果显示,莫诺苷可有效抑制H2O2诱导的细胞内ROS蓄积。6.脂质氧化检测结果表明,莫诺苷可抑制细胞内的脂质发生过氧化;SOD的检测表明,莫诺苷可显著抑制H2O2诱导的SOD含量的下降。7.JC-1荧光探针和分光光度法的结果显示,莫诺苷可显著抑制H2O2诱导的SK-N-SH细胞线粒体膜电位的下降以及Caspase-3的激活。8.Western blot和RT-PCR的结果显示,莫诺苷可有效抑制H2O2诱导的BAX的表达上调,并促进Bcl-2的表达。结论:莫诺苷可抑制H2O2诱导的SK-N-SH细胞氧化损伤,其机制可能与抑制细胞内氧化应激、抑制H2O2诱导的BAX的表达上调,并促进Bcl-2的表达、阻断线粒体凋亡途径密切相关。
[Abstract]:Aim: to establish the oxidative damage model of SK-N-SH cell line hydrogen peroxide-H _ 2O _ 2 and to explore the protective effect of monoside on cell oxidative damage. Methods: SK-N-SH1 cells of human neuroblastoma in logarithmic phase were used. The cells were pretreated with different concentrations of Monoside 1 渭 M 10 渭 M and 100 渭 M for 24 h, and added H2O2(200-400 渭 M for 24 h to induce cell injury. (2) the growth and morphological changes of SK-N-SH cells in each group were observed under inverted phase contrast microscope. The survival rate of SK-N-SH cells was measured by MTT method. Lactic dehydrogenase (LDH) cytotoxicity assay kit was used to detect the amount of LDH released in the cells, and the lipid oxidation assay kit was used to detect the lipid oxidation level of the cells. Superoxide dismutase (SOD) assay kit was used to detect the content of SOD in cells. (4) reactive oxygen species (Ros) assay kit was used to detect the production of ROS in cells. Superoxide anion fluorescence probe (Dihydroethidium) was used to detect the level of superoxide anion in cells. The mitochondrial membrane potential detection kit (JC-1) was used to detect the changes of mitochondrial membrane potential (蠄 m). Caspase-3 activity in cells was detected by spectrophotometry.) apoptosis was analyzed by flow cytometry (FCM) Hoechst staining. The expression of apoptosis-related protein was detected by Western blot. 8) the expression of apoptosis-related genes was detected by RT-PCR. Results: 1. The results of cell viability test showed that H _ 2O _ 2 could decrease the activity of SK-N-SH cells in a dose-dependent and time-dependent manner. After treatment with H2O2(200 渭 M for 24 h, the cell viability was significantly lower than that in the control group, while Monoside could significantly inhibit the decrease of SK-N-SH cell viability induced by H2O2. The morphological results showed that after treatment with H2O2(200 渭 M for 24 h, the SK-N-SH cells showed obvious morphological changes, such as the disappearance of the processes, the swelling and contraction of the cell bodies, the aggregation of some cells and the fragmentation of the fragments of the SK-N-SH cells. The results of cytotoxicity test showed that monoside could reduce the release of intracellular enzyme LDH. 4. The results of flow cytometry and Hoechst staining showed that Monoside could significantly inhibit the apoptosis of SK-N-SH cells induced by H2O2. 5. DCFH-DA and Dihydroethidium fluorescence probe assay showed that Monoside could effectively inhibit the intracellular ROS accumulation induced by H2O2. The results of lipid oxidation test showed that Monoside could inhibit lipid peroxidation in cells. The results showed that Monoside could significantly inhibit the decrease of SOD content induced by H2O2. 7. 7. JC-1 fluorescence probe and spectrophotometry showed that Monoside could inhibit the decrease of SOD content. Monoside could significantly inhibit the decrease of mitochondrial membrane potential induced by H2O2 and the activation of Caspase-3. 8. The results of Western blot and RT-PCR showed that Monoside could effectively inhibit the up-regulation of BAX expression induced by H2O2 and promote the expression of Bcl-2. Conclusion: Monoside can inhibit the oxidative damage of SK-N-SH cells induced by H2O2, and its mechanism may be related to the inhibition of intracellular oxidative stress, the up-regulation of BAX expression induced by H2O2, the promotion of Bcl-2 expression and the blocking of mitochondrial apoptosis pathway.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446

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