- 學位論文
氯離子運輸對於以視網酸誘導P19細胞分化成神經元的影響
The Role of Cl- Transport In Retinoic Acid-Induced P19 Neuron Differentiation
摘要
神經細胞的分化發展是一個相當複雜的過程，包括在各個步驟基因表現的調節。在未成熟的神經細胞，細胞內的氯離子濃度較高，當啟動由γ-丁氨基酪酸 (GABA) 及甘氨酸 (glycine) 所控制的氯離子通道時，會造成細胞膜去極化，導致由電壓門控的鈣離子通道 (voltage-dependent Ca2+ channels；VDCCs) 活化而開啟，使鈣離子進入到細胞內，因此啟動與鈣離子有關的訊息傳遞，這對於神經的分化、生長及成熟扮演關鍵性的角色。另外，有研究指出細胞內pH值 (pHi) 的調節在哺乳類動物生長及發育過程是重要的。而過去對氯-碳酸氫根交換器 (Cl-/HCO3- exchanger；AE) 的研究中顯示，AE的活化對於著床前的胚胎發育時期細胞內的pH值維持是必要的，而AE也牽涉到氯離子的運輸。無論如何，直到目前，氯離子運輸器對於神經分化的影響尚未被釐清。本研究首先探討在以視網酸 (retinoic acid) 誘導P19細胞分化，同時給予氯離子運輸器 (chloride transporter) 的抑制劑4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) 對於神經細胞分化的影響。我們利用神經分化的標記基因class III β tubulin及神經幹細胞未分化的標記基因Oct-3/4來呈現P19細胞神經分化的程度。比較單純以RA (500nM) 誘導及以RA (500nM) 合併DIDS (200μM) 誘導的組別，後者確實顯著增加class III β tubulin mRNA 及蛋白質的表現，而Oct-3/4的表現則是降低。另外在免疫螢光染色的實驗其結果也相同。同時給予氯離子通道 (Cl- channels) 的抑制劑5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) 與RA，亦發現以RA合併NPPB誘導確實顯著增加class III β tubulin 蛋白質之表現。我們也改變細胞外pH值 (pHo)，發現在酸性環境下RA誘導的P19細胞class III β tubulin 蛋白質顯著增加。另外，實驗結果顯示P19細胞表現AE蛋白質亞型AE3。本研究的結論是，抑制氯離子通道及透過酸化刺激AE3，均能加速細胞內氯離子累積，進而促進視網酸誘導P19分化成神經細胞。
並列摘要
The differentiation of mammalian neurons during development is a highly complex process involving regulation and coordination of gene expression at multiple steps. In immature neurons, [Cl−]i is high. The activation of GABA- or glycine-gated Cl− conductances results in membrane depolarization. This lead to activation of voltage-dependent Ca2+ channels (VDCCs) and [Ca2+]i transients that have a central role in neuronal differentiation, growth and maturation. In addition, intracellular pH (pHi) regulates many processes important for cell growth and differentiation. Cl-/HCO3- exchanger (AE) activity is necessary for maintaining pHi in embryonic cells. AE is also responsible for Cl- influx. However, the role of Cl- transporter during neuronal differentiation is not clear. We examined the effect of Cl- transport inhibitor 4,4'-diisothiocyana- tostilbene-2,2'-disulphonic acid (DIDS) on the neuronal differentiation of P19 cells. We used class III β tubulin as the differentiated neuron marker and Oct-3/4 as the undifferentiation stem cell marker. In comparison with RA (500nM) only treatment, combined treatment of RA (500nM) and DIDS (200μM) significantly increased the expression of class III β tubulin while decreased the expression of Oct-3/4, revealed by immunoblotting, real-time PCR and immunofluorochemistry. Another Cl- channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) was also tested. Our experiment showed that combined treatment of RA (500nM) and NPPB (50μM) significantly increased the expression of class III β tubulin. We further examined the effect of changing pHo during the neuronal differentiation of P19 cells. We found that treatment of RA in low pHo significantly increased the expression of class III β tubulin. P19 cells express Cl-/HCO3- exchanger isoform protein AE3. Our results suggest that the inhibition of Cl- channel and the stimulation of AE3 may cause Cl- accumulation in P19 cells, leading to enhancement of RA-induced P19 neuron differentiation.