中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/56419
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    jsp.display-item.identifier=請使用永久網址來引用或連結此文件: http://ir.cmu.edu.tw/ir/handle/310903500/56419


    题名: 電壓調控鉀離子通道在N2A細胞中由cAMP刺激之神經突出新生所扮演的角色
    Voltage-Gated K+ Channels Play a Role in cAMP-Stimulated Neuritogenesis in Mouse Neuroblastoma N2A Cells
    作者: 黃千芳;Chien-Fang Huang
    贡献者: 神經科學與認知科學研究所碩士班
    关键词: 神經突出;神經元;電壓調控鉀離子通道;cAMP;Neurite;Neuron;Voltage-gated K+ channel;cAMP
    日期: 2010-07-29
    上传时间: 2016-01-08 15:23:58 (UTC+8)
    出版者: 中國醫藥大學
    摘要: 神經突出新生在神經迴路的形成過程中佔有重要之角色，因此了解神經突出新生之分子機轉是必要的。神經生長因子與多種因子皆可刺激神經突起生長，目前以增加細胞內第二傳遞訊息cAMP之濃度來刺激神經突出新生是廣泛被使用。已知cAMP-依賴性蛋白質激?? (cAMP-dependent protein kinase A; PKA)可以活化微小管連結蛋白 (microtubule-associated proteins; MAPs)，以幫助微小管成束或是幫助微小管與肌動蛋白 (actin)形成交互作用而刺激神經突出生長。
    本研究證明使用Forskolin和Isobutylmethylxanthine來增加細胞內cAMP之濃度是經由PKA路徑而非胞外調節激?? (extracellular signal-related kinase; ERK)及 p38絲裂原活化蛋白激?? (p38 mitogen- activated protein kinase; p38 MAP kinase)來刺激小鼠神經纖維母瘤細胞的神經突出新生。此外兩個鉀離子阻斷劑4-aminopyridine (4-AP)和Tetraethylammonium (TEA)皆可抑制神經突出之生長，且此兩鉀離子阻斷劑抑制鉀電流與神經突出生長之濃度依賴相關性非常吻合。為了更加證實電壓調控鉀離子通道之角色，我們使用小片段干擾RNA (siRNA)來抑制細胞電壓調控鉀離子通道1.1、1.4及2.1三亞型之基因表現，發現可減少鉀電流外流及神經突出新生。此外已分化之N2A細胞其鉀電流為未分化之細胞的1.6倍，且主要為Kv1.1及Kv1.4兩亞型的增加。於免疫螢光染色下也發現無論是在神經細胞本體或其突出皆有電壓調控鉀離子通道之三亞型之表現。使用PBFI (Potassium-binding benzofuran isophthalate)螢光比值代表細胞內鉀離子濃度時發現，已分化的N2A細胞之神經突出之鉀離子濃度低於神經細胞本體。綜合以上結果，可以確定電壓門控鉀離子通道可以調控以cAMP/PKA刺激小鼠神經纖維母瘤細胞之神經突出新生。
    Neuritogenesis is essential in establishing the neuronal circuitry. One of the most well established intracellular signal causing neuritogenesis is cAMP. cAMP-dependent protein kinase A (PKA) is believed to activate microtubule-associated proteins (MAPs), which then cause neuritogenesis by driving actin and microtubule polymerization. In this report, we showed that an increase in intracellular cAMP triggered by forskolin and isobutylmethylxanthine stimulated neuritogenesis in neuroblastoma N2A cells via a PKA-dependent pathway, but not via extracellular signal-related kinase (ERK). Two voltage-gated K+ (Kv) channel blockers, 4-aminopyridine (4-AP) and tetraethylammonium (TEA), concentration-dependently inhibited neuritogenesis. Such concentration-dependence correlated very well with the concentration-dependence of the two blockers to inhibit Kv currents in N2A cells. In support of this, transfection with specific siRNA knocked down Kv1.1, Kv1.4 and Kv2.1 gene expression, reduced Kv currents and inhibited neuritogenesis. In addition, Kv currents were enhanced by about 1.6-fold in neurite-bearing N2A cells, which could be due to up-regulation of Kv1.1 and Kv1.4. Immunohistochemical staining indicated that Kv1.1, Kv1.4 and Kv2.1 channels all expressed in the cell bodies and neurites. Ratio microfluorimetric imaging of intracellular [K+] using PBFI revealed that [K+] inside the neurite was lower than that inside the cell body. Taken together, the results suggest a role of Kv channels and enhanced K+ efflux in cAMP/PKA-stimulated neuritogenesis in N2A cells.
    显示于类别:[神經科學與認知科學研究所] 博碩士論文

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