氯離子通道ClC-2在以視網酸誘導P19細胞神經分化過程中之角色

Role of Chloride Channel ClC-2 in Retinoic Acid-Induced P19 Neuronal Differentiation

汪維平(Wei-Ping Wang)

指導教授 ： 蔡克勵

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2011年)

摘要

在過去的研究中發現，細胞內氯離子濃度對神經發育有重要的影響，所以我們有興趣了解氯離子通道參與神經分化的過程。我們使用P19細胞作為實驗模型，在視網酸誘導P19神經細胞分化的過程中， class Ⅲ tubulin神經標記基因及蛋白會隨著神經分化而增加。
我們使用不同的氯離子通道抑制劑，抑制不同種的氯離子通道，包括N-苯基胺茴酸、9-蔥羧酸、尼福密酸等試劑，以了解氯離子通道對於神經分化的影響。首先使用琥珀酸去氫酶測定劑，測定P19細胞之存活率，並找出對細胞低毒性的劑量範圍。接下來我們使用這些低毒性的範圍來抑制氯離子通道，結果指出電壓門控的氯離子通道抑制劑N-苯基胺茴酸，對於神經分化有顯著的抑制效果。另外，在P19神經細胞分化過程中，也有電壓門控氯離子通道ClC-1及ClC-2基因及蛋白的表現，其中ClC-1通道蛋白表現量會隨著時間而增加，而ClC-2通道蛋白表現量會隨著時間而降低。我們使用ClC-1及ClC-2基因緘默的方式，偵測之後神經分化受抑制的程度，結果顯示，ClC-2基因緘默組神經標記蛋白的表現量比控制組有顯著下降，可知電壓門控氯離子通道ClC-2在神經分化過程扮演重要的角色。
成熟的神經細胞上會有電壓門控鈣離子通道，在去極化刺激之下會活化，造成鈣離子流入，我們以此作為神經成熟的指標。我們使用離子影像暨活體灌流系統，偵測P19細胞經高鉀溶液去極化後，鈣離子的上升反應。其中我們發現鈣離子反應的程度會隨著神經的成熟而增加，另外，使用N-苯基胺茴酸抑制的組別，鈣離子反應的程度也會比控制組低。
總結以上的實驗，我們證實了電壓門控氯離子通道ClC-2在神經分化過程當中，扮演著必需的角色。

關鍵字

氯離子通道 ； ClC-2 ； 神經分化

並列摘要

It has been shown that intracellular Cl- concentration plays an important role in neuronal differentiation. We investigated the role of voltage-gated chloride channels ClC-1 and ClC-2 during neuronal development. Neuron marker classⅢ ?? tubulin gene and protein were expressed in retinoic acid (RA) -induced P19 neuron differentiation. We used 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay to choose appropriate concentration range of chloride channel inhibitors including anthracene-9-carboxylic acid (9-AC), niflumic acid (NA), and diphenylamine-2-carboxylic acid (DPC). After the treatment of low toxicity dose of voltage-gated chloride channel inhibitors, classⅢ β tubulin protein expression was significantly lowered than control group. The genes of ClC-1 and ClC-2 were also expressed during P19 neuron differentiation. ClC-1 protein expression was increased, but ClC-2 protein expression was decreased during the process. Furthermore, knock down of ClC-2 gene by specific siRNA suppressed P19 neuron differentiation. Because the mature neuron has voltage-gated Ca2+ channels, we measured Ca2+ influx evoked by high K+ treatment in P19 cells to evaluate their differentiation. The Ca2+ influx stimulated by membrane depolarization was also reduced after inhibition of both channels by DPC. We propose that voltage-gated chloride channel ClC-2 is essential in neuronal differentiation.

並列關鍵字

chloride channels ； ClC-2 ； neuronal differentiation

參考文獻

1 Aguado, F. et al. BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl- co-transporter KCC2. Development (Cambridge, England) 130, 1267-1280 (2003).

2 Akerman, C. J. & Cline, H. T. Depolarizing GABAergic conductances regulate the balance of excitation to inhibition in the developing retinotectal circuit in vivo. J Neurosci 26, 5117-5130 (2006).

3 Almaca, J. et al. TMEM16 proteins produce volume-regulated chloride currents that are reduced in mice lacking TMEM16A. The Journal of biological chemistry 284, 28571-28578 (2009).

4 Arreola, J. et al. Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl- channel gene. The Journal of physiology 545, 207-216 (2002).

5 Barish, M. E. A transient calcium-dependent chloride current in the immature Xenopus oocyte. The Journal of physiology 342, 309-325 (1983).