线粒体在PBDE-47致SH-SY5Y细胞凋亡中的作用

发布时间：2018-01-18 15:26

本文关键词：线粒体在PBDE-47致SH-SY5Y细胞凋亡中的作用　出处：《华中科技大学》2013年硕士论文　论文类型：学位论文

【摘要】：多溴联苯醚（Polybrominated diphenyl ethers，PBDEs）的神经毒性作用近年来受到人们的广泛关注，但其具体机制还不十分清楚。本实验通过PBDE-47作用于人神经母细胞瘤细胞（SH-SY5Y细胞），观察其对细胞凋亡率、线粒体膜电位（ΔΨm）、线粒体Ca~(2+)浓度（[Ca~(2+)]m）以及细胞色素C的影响，探讨线粒体在PBDE-47致SH-SY5Y细胞凋亡中的作用，为PBDEs的神经毒性作用机制研究提供理论依据。第一部分PBDE-47对SH-SY5Y细胞凋亡率、线粒体膜电位和线粒体Ca~(2+)浓度的影响目的：研究不同剂量PBDE-47对体外培养的SH-SY5Y细胞凋亡率、线粒体膜电位和线粒体Ca~(2+)浓度的影响。方法：利用线粒体膜电位（JC-1）和线粒体钙离子（Rhod-2/AM）检测荧光探针处理细胞，分别采用流式细胞术（Flow cytometry,FCM）和荧光酶标仪检测PBDE-47作用对SH-SY5Y细胞凋亡率、ΔΨm和[Ca~(2+)]m的影响。结果：与溶剂对照组比较，10μmol/L PBDE-47染毒组的细胞早期凋亡率、总凋亡率明显升高（P 0.05）；10μmol/L PBDE-47染毒组ΔΨm和[Ca~(2+)]m显著下降（P 0.05）。结论：PBDE-47导致ΔΨm和[Ca~(2+)]m下降，可能与其诱导细胞凋亡相关。第二部分PBDE-47对SH-SY5Y细胞内细胞色素C mRNA和蛋白表达的影响目的：探讨PBDE-47对细胞色素C mRNA和蛋白表达水平的影响。方法：提取胞浆RNA利用逆转录PCR法检测胞浆细胞色素C的mRNA表达水平；分离、提取蛋白后利用Western blot技术检测细胞胞浆和线粒体细胞色素C的蛋白表达水平。结果：与溶剂对照组相比，10μmol/L PBDE-47染毒组胞浆细胞色素C mRNA表达水平明显升高（P 0.05）；1、5、10μmol/L PBDE-47染毒组的线粒体细胞色素C蛋白表达水平显著下降（P 0.05），同时，5、10μmol/L PBDE-47染毒组胞浆内细胞色素C蛋白表达水平显著升高（P 0.05）。结论：PBDE-47可导致细胞色素C从线粒体转移到胞浆，从而激活线粒体信号通路，，诱导细胞凋亡。
[Abstract]:The neurotoxicity of polybrominated diphenyl ethersm (PBDEs) has attracted much attention in recent years. However, the specific mechanism is not very clear. In this experiment, human neuroblastoma cell line SH-SY5Y cells were treated by PBDE-47, and the apoptosis rate of SH-SY5Y cells was observed. Mitochondrial membrane potential, mitochondrial Ca~(2 concentration. [To explore the role of mitochondria in the apoptosis of SH-SY5Y cells induced by PBDE-47. To provide theoretical basis for the study of neurotoxicity mechanism of PBDEs. Effect of PBDE-47 on apoptosis rate, mitochondrial membrane potential and mitochondrial Ca~(2 concentration of SH-SY5Y cells Aim: to study the effects of different doses of PBDE-47 on apoptosis rate, mitochondrial membrane potential and mitochondrial Ca~(2 concentration in cultured SH-SY5Y cells. Methods: mitochondrial membrane potential (JC-1) and mitochondrial calcium ion Rhod-2 / AM were used to detect the fluorescence probe. Flow cytometry (FCM) and fluorescence enzyme labeling (FCM) were used to detect the apoptosis rate, 螖蠄 m, and fluorescence enzyme of SH-SY5Y cells induced by PBDE-47. [Ca~(2)] m. Results: compared with the solvent control group, the early apoptosis rate and the total apoptosis rate in 10 渭 mol/L PBDE-47 group were significantly higher than those in the control group (P 0.05). 螖蠄 m and 螖蠄 m in 10 渭 mol/L PBDE-47 exposed group [Ca~(2)] m decreased significantly (P 0.05). Conclusion\\\% PBDE-47 leads to 螖蠄 m sum. [The decrease of Ca~(2] m may be related to the induction of apoptosis. The effect of PBDE-47 on the expression of cytochrome C mRNA and protein in SH-SY5Y cells Objective: to investigate the effect of PBDE-47 on the expression of cytochrome C mRNA and protein. Methods: the mRNA expression level of cytochrome C was detected by reverse transcription PCR method. The protein expression of cytoplasmic and mitochondrial cytochrome C was detected by Western blot. Results: compared with the solvent control group, the expression of cytochrome C mRNA in 10 渭 mol/L PBDE-47 group was significantly higher than that in the control group (P 0.05). The expression of cytochrome C protein in mitochondria of 10 渭 mol/L PBDE-47 group decreased significantly (P 0.05). Cytochrome C protein expression in cytoplasm of 10 渭 mol/L PBDE-47 group increased significantly (P 0.05). Conclusion\% PBDE-47 can induce cytochrome C transfer from mitochondria to cytoplasm. Thus, mitochondrial signaling pathway was activated and apoptosis was induced.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R363

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