臺灣博碩士論文加值系統

NG108-15 細胞株是由 N18TG-2 與 C6-BU-1 融合而得，常用來作為研究神經發育以及神經分化的良好模式。本論文發現，在只有1% 血清存在下會抑制細胞生長，並以 1mM dibutyryl cAMP (dbcAMP) 處理細胞，提升胞內 cyclic adenosine 3',5'-monophosphate (cAMP) 的濃度，可透過活化 extracellular-signal-regulated kinase (ERK) 及cAMP response element binding protein (CREB) 而使 NG108-15 細胞分化。分化特徵包括 (1) 神經突、神經節的形成及細胞體轉趨圓形 (2) 電感性鈣離子通道 (voltage sensitive calcium channel, VSCC) 活性增加 (3) 神經細胞指標蛋白 Microtubule Associated Protein 2 (MAP2) 的表現增加。Rapamycin 會抑制 Mammalian Target of Rapamycin Complex 1 (mTORC1) 活性，mTOR 是控制細胞週期由 G1 進入 S 期的蛋白激脢，其能促進細胞生長且抑制細胞自噬 (autophagy)，所以 rapamycin 能促使細胞自噬，及將細胞週期停滯在 G0/G1 。在給予 dbcAMP 同時再給予 rapamycin，NG108-15 細胞之分化較單獨處理 dbcAMP 更早發生：神經節及神經突的數目更多，VSCC 活性更強，MAP2 在處理2天後即表現；以 siRNA 技術抑制 mTOR 蛋白表現，亦可觀察到相似的結果；此外，在有 dbcAMP 及 rapamycin 存在下，以細胞自噬抑制劑或以 siRNA 技術抑制 Beclin1 之表現，均能有效抑制細胞自噬，也均能反轉 dbcAMP 及 rapamycin 同時存在下所誘發的早發性分化；單獨處理 rapamycin 可以誘發些許的分化作用；rapamycin 不會強化 dbcAMP 誘發的 ERK 及 CREB 之磷酸化，而 dbcAMP 也不會誘發細胞自噬。所以，以 rapamycin 或以 siRNA 技術抑制 mTOR增加細胞自噬，加上 dbcAMP 誘發的 ERK 及CREB 磷酸化，能加速 NG108-15 細胞之分化。

NG108-15 cell line fused from N18TG-2 and C6-BU-1 is a good model for investgating neuronal development and differentiation. We found that cell proliferation would be inhibited by treating DMEM with only 1% serum. It has been already known that inhibiting cell proliferation and elevating concentration of cytosolic cyclic adenosine 3',5'-monophosphate (cAMP) would let NG108-15 cell differentiation through activating transcription factor, cAMP response element binding protein (CREB). There are three features when NG108-15 cell differentiate. (1) Neurite outgrowth and Formation of varicosity. (2) Activity of voltage sensitive calcium channel, VSCC, elevates. (3) Neuronal marker protein, Microtubule Associated Protein 2 (MAP2), formate. Rapamycin is an inhibitor of Mammalian Target of Rapamycin Complex 1 (mTORC1). mTOR is a protein kinase that control cell cycle from G1 to S phase, promote proliferation and inhibit autophagy. Therefore, rapamycin would arrest cell cycle in G0/G1 and promote autophagy. We found that treating dibutyryl cAMP (dbcAMP) and rapamycin at the same time would promote NG108-15 cell differentiation earlier than only treating dbcAMP. It would have higher neurite and varicosity number, VSCC activity and MAP2 content. Silencing mTOR would mimic the effect of rapamycin in NG108-15 cells. Furthermore, potentiating differentiation caused by cotreating dbcAMP and rapamycin would be inhibited by adding autophagic inhibitor or silencing Beclin1. Rapamycin could also induce differentiation in NG108-15 cells. Rapamycin would not change phosphorylatic level of ERK and CREB. Besides, dbcAMP would not induce autophagy. In sum, differentiation of NG108-15 cells would be potentiated through inducing autophagy by treating rapamycin or silencing mTOR.

正文目錄 I
圖文目錄 II
縮寫表 III
中文摘要 IV
英文摘要 VI
緒論 1
實驗材料與方法 7
實驗結果 22
討論 37
結論 42
參考文獻 58

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