水溶性CoQ10对鱼藤酮诱导PC12细胞线粒体动态平衡及细胞凋亡的保护机制研究

发布时间：2018-03-08 19:20

  本文选题：水溶性辅酶Q10　切入点：鱼藤酮　出处：《宁夏医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的探讨水溶性辅酶Q10(CoQ10)对鱼藤酮(Rotenone,Rot)诱导帕金森病(PD)细胞模型细胞凋亡的保护作用,以及改善线粒体功能障碍及线粒体融合裂解动态失衡的机制研究,为水溶性CoQ10用于PD的治疗提供理论依据。研究方法1.采用已经由神经生长因子诱导分化的PC12细胞传代培养,选取合适的Rot浓度处理对数生长期的PC12细胞建立PD细胞模型。2.确定水溶性CoQ10浓度作为治疗浓度,将实验分为溶剂对照(vehicle组)组,Rot组,CoQ10组和CoQ10治疗组。3.cck-8法检测细胞活性,并在倒置显微镜下观察细胞形态。4.ROS检测试剂盒检测各组细胞内ROS变化。5.JC1-Mitochondrial膜Potential Assay试剂盒染色后使用流式细胞仪分析各组线粒体膜电位(MMP)变化。6.MitoTracker线粒体荧光探针标记线粒体,激光共聚焦显微镜观察线粒体形态变化。7.Western blotting检测各组细胞凋亡以及线粒体融合分裂相关蛋白表达变化。结果1.Rot处理24h后的PC12细胞存活率显著降低并与剂量呈负相关,CoQ10可改善Rot处理诱导的PC12细胞存活率降低。2.Rot引起ROS水平升高,CoQ10可降低Rot处理后细胞内的ROS水平。3.Rot诱导PC12细胞线粒体膜电位(MMP)降低,导致线粒体片段化,水溶性CoQ10可改善Rot处理诱导的PC12细胞MMP降低和线粒体片段化。4.Western blotting实验表明CoQ10可降低Rot引起的Caspase-9、active Caspase-3以及Bax的表达增多,上调Bcl-2的表达,阻止AIF向核内转移;水溶性CoQ10可降低Rot引起的线粒体分裂蛋白Drp1表达增多,上调线粒体融合蛋白Mfn2、OPA1的表达,对Mfn1、Fis1的表达无明显影响。结论1.水溶性CoQ10可改善Rot致PC12细胞MMP降低及线粒体片段化。2.水溶性CoQ10对Rot致PC12细胞凋亡具有保护作用。其机制可能是通过降低细胞氧化应激以及线粒体通路细胞凋亡实现的。3.水溶性CoQ10对Rot致PC12细胞线粒体融合、分裂紊乱具有保护作用。其机制可能是通过上调Mfn2、OPA1的表达,下调Drp1的表达实现。
[Abstract]:Objective to investigate the protective effect of water-soluble coenzyme Q10 CoQ10 on apoptosis induced by rotenonerotenone rotator (rotenonerotenone Rot) and to investigate the mechanism of improving mitochondrial dysfunction and dynamic imbalance of mitochondrial fusion cleavage. To provide theoretical basis for the treatment of PD with water-soluble CoQ10. Methods 1. PC12 cells, which have been induced by nerve growth factor (NGF), were subcultured. PD cell model was established by selecting appropriate Rot concentration to treat PC12 cells in logarithmic growth phase. 2. The concentration of water-soluble CoQ10 was determined as the therapeutic concentration. The experiment was divided into two groups: Rot group, Rot group, CoQ10 group and CoQ10 treatment group, and the cell activity was detected by the method of 3.cck-8. Cell morphology was observed under inverted microscope. 4. Ros assay kit was used to detect the changes of intracellular ROS. 5. JC1-Mitochondrial membrane Potential Assay kit was stained. The changes of mitochondrial membrane potential were analyzed by flow cytometry. 6. MitoTracker mitochondrial fluorescence probe was used to label mitochondria. The morphologic changes of mitochondria were observed by laser confocal microscope. 7. Western blotting was used to detect apoptosis and the expression of mitochondrial fusion mitogen-associated protein. 1. The survival rate of PC12 cells after 24 hours of Rot treatment decreased significantly and showed negative phase with dose. 2. CoQ10 can improve the survival rate of PC12 cells induced by Rot. 2.Rot induced increase in ROS level. CoQ10 can reduce the ROS level of PC12 cells after Rot treatment. 3. Rot induced mitochondrial membrane potential of PC12 cells. Water soluble CoQ10 could improve the MMP reduction and mitochondrial fragmentation of PC12 cells induced by Rot. 4. Western blotting experiment showed that CoQ10 could decrease the expression of Caspase-9 active Caspase-3 and Bax induced by Rot, upregulate the expression of Bcl-2 and prevent AIF from transferring to nucleus. Water soluble CoQ10 could decrease the expression of mitochondrial mitogen Drp1 induced by Rot, and up-regulate the expression of Mfn2OPA1, a mtDNA fusion protein. Conclusion 1. Water soluble CoQ10 can improve the decrease of MMP and mitochondrial fragmentation in PC12 cells induced by Rot. 2. Water-soluble CoQ10 can protect PC12 cells from apoptosis induced by Rot. The mechanism may be by reducing cell oxidation. 2. Stress and apoptosis of mitochondrial pathway. 3. Rot induced mitochondrial fusion of PC12 cells by water-soluble CoQ10. The mechanism may be by up-regulating the expression of MfN2 OPA1 and down-regulating the expression of Drp1.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5

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