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研究生:黃瑋琪
研究生(外文):Huang wei-chi
論文名稱:腺嘌呤核苷三磷酸接受器調控RBA-2第二型星狀膠細胞內麩氨合成酵素之表現
論文名稱(外文):Involvement of ATP and purinergic receptor on glutamine synthetase in RBA-2 astrocytes
指導教授:孫興祥
指導教授(外文):Synthia H. Sun
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:60
中文關鍵詞:星狀膠細胞麩氨合成酵素
外文關鍵詞:astrocytesglutamine synthetase
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星狀神經膠細胞 (Astrocyte) 具有一個重要的功能,就是將突觸間的麩氨酸 (glutamate) 清除,目的為了避免過多的麩氨酸造成興奮性神經毒性 (excitatory neurotoxicity),而這些回收回來的麩氨酸就是透過麩氨合成酵素 (Glutamine Synthetase) 這個酵素轉變成為麩醯胺酸(glutamine)。目前的研究已經證實麩氨合成酵素在一些神經退化性疾病或腦部受傷缺氧所引起的神經毒性中扮演一個重要的角色。但是在腦部受傷時,調控麩氨合成酵素的分子機轉仍不清楚。
從目前的研究我們發現,第二型星狀膠細胞--RBA-2,在正常的細胞培養情況下,麩氨合成酵素的活性及蛋白質表現量遠比C6神經膠瘤細胞株和IA1g1星狀神經膠細胞株多。另外也發現外加麩氨酸會增加麩氨合成酵素的活性和蛋白質的表現。在高濃度的ATP(1mM)刺激之下,發現隨時間的增加,麩氨合成酵素的活性和蛋白質的表現都會下降。為了探討P2X7接受器的活化是否有參與此訊號傳遞,我使用了P2X7接受器的agonist,BzATP和antagonist,oATP等藥物觀察是否影響ATP對麩氨合成酵素的作用,結果發現oATP抑制了ATP和BzATP引起的麩氨合成酵素蛋白質表現的下降。因為已經知道活化P2X7接受器後會造成細胞外鈣離子流入細胞內以及PKC的活化,結果也發現在沒有鈣離子的存在下,ATP對麩氨合成酵素蛋白質的表現的作用受到抑制;加入PKC的抑制劑也會抑制ATP對麩氨合成酵素蛋白質表現的作用。另外,為了探討c-Jun是否有參與在這個訊號傳遞的過程中,我使用了活化c-Jun的kinase─JNK (c-Jun N-terminal kinase) 的抑制劑--- JNK inhibitor Ⅰ,發現處理JNK inhibitor Ⅰ後無法完全抑制ATP對麩氨合成酵素的作用,表示c-Jun不參與ATP所造成的麩氨合成酵素活性和蛋白質表現的下降。
綜合以上的結果,我們發現RBA-2有豐富的麩氨合成酵素,ATP使麩氨合成酵素的活性和蛋白質的表現下降,是需要透過P2X7接受器的活化,以及細胞外鈣離子流入細胞內,也證實PKC的活化有參與其中。
One of the important physiological functions of astrocyte is to metabolize excitatory neurotransmitter, glutamate. Therefore, astrocytes play a key role in protection of neurons from ischemia-induced excitatory toxicity through taking up excess glutamate and converting it to glutamine via the enzyme glutamine synthetase. Recent studies have demonstrated that glutamine synthetase may be a critical factor in the neurotoxicity produced after CNS injury or ischemia. However, the molecular mechanisms that modulate GS expression in astrocytes after CNS injury are not clear.
The study investigates the effect of ATP on glutamine synthetase activity and protein expression of RBA-2 type 2 astrocytes. RBA-2 astrocytes expressed abundant glutamine synthetase in the basal culture condition as compared with C6 astrocytoma cells and IA1g1 type-1 astrocytes. Exogenous glutamate induced a dose-dependent increase in GS activity and protein expression. Treating the cells with ATP (1 mM) caused a significant decrease on GS activity and protein expression . Because RBA-2 astrocytes were known to express P2X7 receptor, and activation of P2X7 receptor stimulated Ca2+ influx and protein kinase C , we elucidate whether activation of P2X7 receptors and the signal mechanisms are involved in the ATP-altered GS expression. The P2X7 receptor selective antagonist, oATP ( 100 μM ) inhibited the ATP- and BzATP-reduced GS expression. Treatment of cells with Ca+2 free buffer inhibited the ATP- and BzATP-reduced GS expression. In addition, treatment of cells with PKC inhibitors GF109203X and GÖ6976 rescued the ATP- and BzATP-reduced GS expression. Because c-jun was known to mediate glutamine synthetase regulation, RBA-2 was treated with JNK inhibitor Ⅰ. The result showed that JNK inhibitor Ⅰcan’t inhibited the effect of ATP on the glutamine synthetase expression.
Take together, we have demonstrated that RBA-2 expressed abundant GS, glutamate enhanced GS expression, and exposure of astrocyte to ATP altered GS activity and protein expression. The decrease in GS protein expression is dependent on PKC activation via P2X7 receptors.
頁次
英文縮寫對照表.................................1
中文摘要.......................................2
英文摘要.......................................4
序論...........................................6
實驗材料與方法.................................13
結果...........................................20
討論...........................................27
參考文獻.......................................33
圖表...........................................44
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