研究鈣離子在神經幹細胞分化過程中的角色

神經幹細胞為一具自我更新和多重分化能力之原母細胞，而在調控神經幹細胞分化上其機制還尚未明確瞭解。研究發現，細胞內鈣離子的濃度變化可調控許多細胞內生理功能，例如：神經傳導、肌肉收縮、細胞增生及分化。而在過去研究證實dibutyryl cAMP (dbcAMP) 可誘發神經幹細胞的分化，並引起胞外鈣內流的現象產生。我們主要利用鈣離子攜帶者離子黴素，藉以瞭解單獨引發胞內鈣離子增加是否可誘發神經幹細胞的分化。在本篇研究將探討離子黴素在神經幹細胞分化上引起的作用和其活化之訊息傳遞路徑。離子黴素誘發之分化情形可藉由神經突長的產生、神經元特有離子通道VOC之表現和神經元特殊蛋白增加等方面來測定。結果顯示，神經幹細胞經離子黴素處理後，型態上並未有神經突長現象的產生。在測定神經幹細胞是否具備功能上，則藉由高鉀溶液刺激後，觀察細胞內鈣離子濃度是否改變來判定。結果顯示，神經幹細胞處理 ionomycin 後，並無法產生與已分化且具神經特性之神經幹細胞類似的胞內鈣離子反應。而以流式細胞儀和西方墨點法分析顯示，離子黴素可刺激細胞早期分化，使神經幹細胞表現神經特有蛋白MAP2、TUJ1。其次，預先處理不同的藥理性抑制劑 FK-506 (calcineurin 抑制劑) 和 PD98059 (MEK 抑制劑) 後顯示，PD98059 可抑制 dbcAMP 所誘發之TUJ1表現；相反地，以離子黴素處理之神經幹細胞則否。值得注意地，若兩者皆處理 calcineurin 抑制劑後，TUJ1的表現量皆會受到抑制。此外，我們發現當神經幹細胞處理離子黴素或 dbcAMP 後，皆可發現兩者的磷酸化 Akt 表現增加。總結以上結果顯示，胞內鈣離子濃度的上升參與了初期的神經幹細胞分化，且可能透過calcineurin之訊息傳遞路徑，但此調控機制仍不足以誘發神經幹細胞分化為成熟的神經細胞。

關鍵字

鈣離子；神經幹細胞；分化

並列摘要

Neural stem cells (NSCs) are defined as cells with self-renewal and multi-potential capability. However, little is known about the mechanisms that regulate differentiation of NSCs. It has been reported that many kinds of physiological functions, including neural conduction, muscle contraction, cell proliferation and differentiation could be controlled by intracellular calcium concentration. We recently demonstrated that dbcAMP can induce differentiation of NSCs and calcium influx. To check whether only cytosolic calcium increase can induce NSCs differentiation, we used calcium ionophore ionomycin. In this study, we determined the effect of ionomycin on NSCs differentiation and examined activation of key signaling pathway. Ionomycin induced differentiation as determined by an increase in neurite outgrowth, increased expression of VOC and neural specific protein. The data showed that ionomycin-induced neural stem cells were not triggered neurite outgrowth. By using intracellular calcium imaging, based on cellular response to KCl, which allows the functional evaluation of neural differentiation. Exposure of NSCs cultures to ionomycin didn’t resulted in increased differentiation of cells displaying a neuronal-like profile of cytosolic calcium response. Using Western blotting and flow cytometry approach to examine the expression of the neuronal marker TUJ1 and MAP2, and the results suggested that exogenous application of ionomycin to cultures of NSCs stimulates their early differentiation leading to increase expression of neuronal markers. In this study, we also pretreated neural stem cells with several potent inhibitors, such as calcineurin inhibitor FK-506 and MEK inhibitor PD98059. PD98059 significantly inhibited TUJ1 expression of dbcAMP-treated, but not ionomycin-treated cells. Significantly, both groups observed that inhibition of calcineurin resulted in decreased expression of TUJ1. Furthermore, we found that exposure of NSCs to both dbcAMP or ionomycin could cause an increase of the phospho-Akt immunoreactivity. Taken together, these results provide initial evidence that intracellular calcium elevation has a role in early neuronal differentiation, and that its effects may be mediated in part by the calcineurin signal transduction pathway. But this regulating system is not sufficient to induce the neuronal maturation.

並列關鍵字

calcineurin ； NSCs ； ionomycin

參考文獻

曹翔凱. 2007. 研究 dbcAMP 在神經幹細胞的分化效果. 私立中原大學化學系碩士論文.

Altman J, Das GD. 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. Journal of Comparative Neurology 124: 319-335.

Alvarez-buylla A, Seri B, Doetsch F. 2002. Indentification of neural stem cells in the adult vertebrate brain. Brain Research Bulletin 57: 751-758.

Berridge MJ. 1998. Neural calcium signaling. Neuron 21: 13-26.

Bolotina VM. 2004. Store-operated channels: diversity and activation mechanisms. Science Signaling 243: e34.

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研究鈣離子在神經幹細胞分化過程中的角色

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