细胞内钾离子稳态参与神经细胞凋亡机制的研究
文献类型：学位论文
| 作者 | 陶艳梅 |
| 学位类别 | 博士 |
| 答辩日期 | 2005 |
| 授予单位 | 中国科学院上海生命科学研究院神经科学研究所 |
| 授予地点 | 中国科学院上海生命科学研究院神经科学研究所 |
| 导师 | 王以政 |
| 关键词 | 神经细胞凋亡 细胞内钾离子浓度 DNA结合 |
| 其他题名 | The action of intracellular potassium on NF-κB/DNA interaction regulates the expression of Bcl-Xs and participates in the neuronal apoptotic process initiated by serum deprivation |
| 中文摘要 | 凋亡细胞内钾离子外流是除胞体皱缩，核固缩，染色体断裂等现象外的细胞凋亡的特征之一。近年来的研究发现细胞胞内钾离子浓度降低，并非以往认为的仅是凋亡进程的一个伴随结果。相反，大量工作显示细胞丢钾是凋亡进行的必要条件。钾通道阻断剂或胞外给予高钾可以阻断多种类型细胞的细胞凋亡。在神经细胞和淋巴细胞上，胞内低钾对细胞凋亡的重要性己有比较翔实的证据。但关于细胞是如何丢钾，以及细胞丢钾是如何促进细胞凋亡进程的两个重要问题仍没有很好的答案。根据丢钾的神经细胞凋亡依赖于新生蛋白合成的事实，我们探讨了胞内钾离子浓度变化与促凋亡蛋白转录调控的关系，试图找出细胞丢钾促进细胞凋亡的胞内信号转导途径。去血清处理原代培养的皮层神经元是已经建立良好的细胞丢钾会促进凋亡的神经细胞模型：已知去血清处理会导致原代培养的皮层神经元胞内丢钾，以广谱钾通道阻断剂四乙基胺（TEA）阻断外向钾电流可以阻断凋亡。这种TEA阻断钾离子外流阻止凋亡的作用不依赖于细胞膜去极化和钙离子内流，而仅仅是因为保持了细胞内的钾离子水平而实现的。利用这个丢钾促进凋亡的细胞模型，我们检测了在神经系统有多种功能的转录因子NF-κB的转录活性受细胞内钾离子浓度变化调控的可能性。通过凝胶阻滞实验（EMSAs），蛋白免疫印迹实验（Westem blotting），免疫荧光细胞化学（Inunnunocytochemistw）和染色质免疫沉淀（Chromatin Immunoprecipitation，ChJP）等方法，我们发现去血清可以导致神经元内NF-κB的抑制蛋白I出。降解，促进NF一B激活，获得DNA结合能力，并转移至核内，与NF-kB调节的靶基因启动子上结合位点结合。以TEA阻止去血．清造成的细胞内钾浓度的改变并不能影响1kBa的降解，NF-kB结合DNA能力的活化，以及NF一出核转移的过程，说明NF-kB的激活和核转移与细胞内丢钾无关。但是去血清诱导的NF-KB与其靶基因启动子上结合位点的实际结合，以及NF-kB下游基因Bcl-Xs，IKBa和kB-tuciferase的转录都能被阻断钾离子外流所抑制。离体EMSA实验证实钾离子浓度的改变可以直接影响NF-KB和DNA之间的相互作用，而不影响NF-kB复合物的亚基之间的相互作用。而且，钾离子对转录因子和DNA之间的这种直接作用具有一选择性，如Forkhead转录因子与DNA的结合同样受钾离子浓度的调节，而SPI，E2F和STAT等转录因子结合DNA的活性刘钾离子浓度变化不敏感。由以上实验结果，我们得出这样的结论，神经细胞内的低钾浓度可以直接促进被激活的转录因子NF-κB与下游基因启动子序列结合位点的结合，从而提高NF-κB下游基因的转录效率。我们进一步利用RNA干扰技术，将NF-κB的主要亚基p65或其下游促凋亡基因Bd-Xs的siRNA转入发育后期的神经元，发现下调p65和Bcl-Xs的蛋白水平可以提高去血清处理后神经元的成活率。而且ChIP和Western blotting的实验结果证实成熟神经元中的Bcl-Xs确实受NF-κB的调节。因此，我们的研究表明NF-κB／Bcl-Xs通路的激活促进成熟神经元凋亡。本研究提出了低钾促进神经细胞凋亡的信号转导途径，提供了全新的细胞内离子强度可能参与细胞内信号转导途径调控的证据，并且证实了在神经细胞凋亡中角色有争议的NF-κB通过转录调控促凋亡蛋白Bd-Xs参与成熟神经元凋亡的进程。 |
| 英文摘要 | The decrease of intracellular potassium concentration ([K~+]_i) is one of the features of cell apoptosis. Recent studies demonstrated that [K~+]_i decrease is not only a consequence of apoptosis, but also the permission for apoptotic progression. Preventing potassium efflux with potassium channel blockers, or high extracellular potassium concentration, protect various cell types from death induced by different insults. The importance of low [K~+]_i to apoptotic process has been convincingly proved in cortical neurons and lymphocytes. However, two questions remain opened: 1, which pathway induces the intracellular potassium loss? 2, how low [K~+]_i promotes apoptotic process? It has been reported that the new protein synthesis is required for the neuronal death related to intracellular potassium loss. Therefore, we investigated whether the alteration of [K~+]_i regulates transcription of apoptotic proteins, in order to discover the signal transduction pathway that intracellular potassium loss promotes apoptosis. Serum deprivation causes cortical neuronal death through elevating the potassium outward current in early phase of cell apoptosis. Blockade of the potassium efflux with tetraethylammonium (TEA), a global potassium channel blocker, prevents cortical neuronal death independently of cellular depolarization or calcium influx. We established this neuronal apoptotic cell model related to intracellular potassium loss in our laboratory, and examined whether the activity of NF-κB, a transcription factor that has been reported involved in neuronal death, was regulated by alteration of [K~+]_i. By using electrophoretic mobility shift assays (EMSAs), Western blotting, Immunocytochemistry and Chromatin immunoprecipitation (ChIP), we demonstrated that serum deprivation caused the degradation of IKBCC (the cytosol inhibitory protein of NF-κB), the possession of DNA-binding ability and the nuclear translocation of NF-κB, and the actually binding to endogenous loci within NF-κB downstream genes in vivo. Preventing intracellular potassium loss by TEA did not affect NF-κB activation and DNA-binding in intact cells induced by serum deprivation, suggesting that those processes were independent of alteration of [K~+]_i. However, preventing [K~+]_i change did suppress the binding of NF-κB to endogenous loci in vivo and the transcription efficiency of NF-κB target genes, Bcl-Xs, IicBa and KB-luciferase. EMSAs in vitro proved that the alteration of potassium concentration affected directly NF-κB/DNA interaction without effect on interaction between the subunits which composed the NF-κB complex. Moreover, the direct action of potassium on the transcription factors/DNA interaction has selectivity, because the DNA binding activity of other neuronal transcription factors, Spl, E2F and STAT were not sensitive to potassium concentration change, although Forkhead transcription factor exhibited the same K+ sensitivity as NF-KB. Taken together, we concluded that the low potassium concentration in neurons induced by serum deprivation helps the binding of NF-KB to its target genes' promoter regions and improves the transcription. We further examined the roles of NF-κB and Bcl-Xs in neuronal death by using RNA interference. We found that down-regulation of p65, the major subunit of NF-KB in cortical neurons, or Bcl-X protein levels survived neurons from serum-deprived treatment. ChIP assays and Western blotting demonstrated that the expression of Bcl-Xs was indeed the consequence of NF-κB activation in mature neurons. Thus, our experiments supported that the activation of NF-κB/BCI-XS pathway promotes the apoptosis of mature neurons. Our study provided the signal transduction pathway how low [K~+]_i- participates in neuronal apoptosis and emphasized the possibility that intracellular ionic strength is involved in regulation of intracellular signal transduction. Furthermore, we proved that NF-κB, which role is controversial in nervous system, is essential to pro-apoptosis in mature neurons through transcription of the pro-apoptotic protein, Bcl-X_s. |
| 语种 | 中文 |
| 公开日期 | 2012-11-26 |
| 页码 | 160 |
| 源URL | [http://ir.sibs.ac.cn/handle/331001/2267]  |
| 专题 | 上海神经科学研究所_神经所(总) |
| 推荐引用方式 GB/T 7714 | 陶艳梅. 细胞内钾离子稳态参与神经细胞凋亡机制的研究[D]. 中国科学院上海生命科学研究院神经科学研究所. 中国科学院上海生命科学研究院神经科学研究所. 2005. |
入库方式： OAI收割
来源：上海神经科学研究所
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