2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/648559摘要：ClC-2是一種藉由膜電位過極化而開啟的氯離子通道，其表現之區域相當廣泛，幾乎遍及所有組織與細胞株。ClC-2氯離子通道在腦神經細胞、上皮細胞、以及神經膠細胞都有相當高的表達程度。雖然其表現之區域相當廣泛，ClC-2氯離子通道之確切腦神經功能為何，至今尚未有定論。因此，本研究計畫的目的是要探討ClC-2氯離子通道之神經生理功能。我們將使用多種不同的實驗技術來釐清ClC-2氯離子通道在腦神經細胞和神經膠細胞中所扮演的生理功能。此外，我們還將嘗試了解ClC-2氯離子通道在成人最常見的惡性腫瘤─神經膠瘤─之潛在病生理角色。茲將本計畫之研究目標概略如下：研究目標一： 建立與測試多個ClC-2氯離子通道改變種。我們將製作多個ClC-2氯離子通道改變種以進行電生理、生化、以及細胞生物測試。此外，我們還將嘗試產生專門認識ClC-2氯離子通道之細胞外結構之抗體，以便爾後研究ClC-2氯離子通道在體外表達細胞和原生細胞之細胞膜表達狀態。研究目標二： 探索腦神經細胞之ClC-2氯離子通道訊息傳遞機制。目前學界對於ClC-2氯離子通道在神經細胞中之蛋白質調控與訊息傳遞機制尚未明瞭。我們將利用酵母雙雜交技術找尋在大鼠腦中與ClC-2氯離子通道有交互作用的蛋白質或因子。研究目標三： 利用核醣核酸干擾技術理解ClC-2氯離子通道之神經生理功能。另外一種探討ClC-2氯離子通道神經生理功能的方式是利用核醣核酸干擾技術來抑制ClC-2氯離子通道在神經細胞的表達。我們將使用離體培養的大腦與海馬迴神經細胞，以研究抑制ClC-2氯離子通道表達對神經興奮性、細胞恆定作用、和神經突觸結構的影響。研究目標四： 評估ClC-2氯離子通道在神經膠和神經膠瘤細胞中所扮演的細胞生理功能。ClC-2氯離子通道在神經膠和神經膠瘤細胞之生理意義為何目前仍然了解不多。我們將聚焦於ClC-2氯離子通道在神經膠細胞之次細胞分佈形態；此外，我們還將嘗試了解ClC-2氯離子通道在神經膠瘤增生現象中之病生理角色。<br> Abstract: ClC-2 is a broadly expressed hyperpolarization-activated Cl- channel, virtually found in all tissues and cell lines investigated. ClC-2 channel is most abundant in brain and epithelia, and is also highly expressed in glial cells. Despite of its ubiquitous expression pattern, the precise neurophysiological role of ClC-2 channels is largely unknown. The purpose of this grant proposal is thus to discern the neurophysiological significance of ClC-2 channels. We will apply multi-disciplinary approaches to explore ClC-2 physiology in both neurons and glial cells. Moreover, we will evaluate the potential contribution of ClC-2 channels in the pathophysiology of gliomas, one of the most frequent and malignant brain tumors in adults.The specific aims of this proposal are:Aim 1: Construction and characterization of various ClC-2 channel constructs. We will generate various ClC-2 constructs for electrophysiological, biochemical and cell biological characterizations. Furthermore, we will attempt to generate specific antibody that recognizes the extracellular domain of ClC2 channel, thereby allowing us to study the surface expression of ClC-2 channels in both heterologous expression systems and native cells.Aim 2: Identification of novel ClC-2 signaling pathways in the brain. There is virtually no information available regarding the protein modulation and signaling mechanism of ClC-2 channels in the brain. We will address this issue by employing yeast-two-hybrid screening to probe for potential ClC-2-binding proteins or ClC-2-modulating factors.Aim 3: Recognition of ClC-2 channel function in neurons by applying the RNAi technology. Another approach to understanding the physiological role of ClC-2 channels in neurons involves suppression of neuronal expression of ClC-2 channels via RNAi technology. By using cultured cortical and hippocampal neurons, we will examine the impact of ClC-2 knock out on membrane excitability, cellular homeostasis, as well as molecular organization of synapses.Aim 4: Evaluation of the cell physiological role of ClC-2 channels in normal glia cells and gliomas. Very little is known about the physiological significance of the expression of ClC-2 channels in glial cells in both normal and cancerous states. Here we will focus on the subcellular localization of ClC-2 channels in glia cells; in addition, we want to know if the expression state of ClC-2 channels may be linked to the pathogenesis of gliomas.氯離子通道 神經細胞 酵母雙雜交技術 訊息傳遞Cl- channel Neuron Yeast-two-hybrid screening Signalling mechanism