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研究生:許書豪
研究生(外文):Shiu, Shu-hau
論文名稱:過氧化氫促使 NG108-15 細胞分化作用
論文名稱(外文):Hydrogen Peroxide Promotes Differentiation Of NG108-15 Hybrid Cells
指導教授:闕小輝
指導教授(外文):Chueh, Sheau-huei
口試委員:劉佩珊王正康
口試日期:2012-06-29
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:33
中文關鍵詞:過氧化氫分化神經突電感性鈣通道細胞自噬細胞凋亡
外文關鍵詞:hydrogen PeroxidedifferentiationneuriteVSCCautophagyapoptosis
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NG108-15 細胞在不同的胞內或胞外因子存在下,不論在形態上或功能上,
均會分化成更具神經細胞特質的細胞。本研究探討 H 2 O 2 是否具促使
NG108-15 細胞分化的能力,細胞以 10µM H 2 O 2 持續處理兩天後,有促分化
的作用,不但細胞的生長靜止,且細胞體會轉趨圓形,有較多神經突 (neurite)
長出,此為典型 NG108-15 細胞分化之特徵,此抑制的生長不是歸因於計劃
性細胞死亡 (apoptotic cell death),也不是歸因於細胞自噬性細胞死亡
(autophagic cell death)。在 10µM H 2 O 2 處理過的細胞,高鉀溶液引起的胞內
鈣上升,較控制組高,表示電感性鈣通道活性較高,此外 Bradykinin (BK)
及 Thapsigargin (TG) 引起的胞內鈣上升,較控制組高,表示 BK-IP 3 的訊號
傳遞途徑,有較高活性,胞內鈣庫儲鈣量也加大。在 H 2 O 2 處理的細胞,在
1 小時後,p38 MAPK 之磷酸化有增加,且持續 24 小時,而 ERK 及 Akt 之
磷酸化不因 H 2 O 2 處理而改變。H 2 O 2 處理後引起的電感性鈣通道活性增加、
鈣庫鈣儲量之增大及 BK-IP 3 訊號傳遞途徑強化,會因 p38 MAPK 抑制劑
SB203580 之加入均被反轉,均與控制組無差異,但 ERK 及 Akt 抑制劑
LY294002 及 PD98059 則對 H 2 O 2 作用無影響。所以綜合我們的結果顯示,在
NG108-15 細胞,低劑量的 H 2 O 2 具促分化之作用,此作用透過活化 p38 MAPK
達成。
Under the influence of various extracellular or intracellular factors,
neuroblastoma × glioma hybrid NG108-15 cells morphologically and functionally
further differentiated into mature neuron. This study examined whether
hydrogen peroxide (H 2 O 2 ) promotes differentiation of NG108-15 cells. After
treatment of cells with 10μM H 2 O 2 , for 2 days, the growth of NG108-15 cells
was halted, the cell bodies were round up and more neurites were elongated,
which are typical features of differentiated cells. High K + induced cytosolic Ca 2+
increase was higher in H 2 O 2 -treated cell than in control cells, suggesting a higher
activity of voltage sensitive Ca 2+ channel in H 2 O 2 treated cells. In addition Ca 2+
increase induced by bradykinin and thapsigargin were also higher in H 2 O 2 -treated
cell suggesting more active bradykinin-IP 3 signaling pathway and greater Ca 2+
stored in Ca 2+ pool , respectively in H 2 O 2 -treated cell. The arreted growth caused
by H 2 O 2 is not attributed to apoptotic and autophagic cell death, while both were
observed by treated cell with thapsigargin. Phosphorylation of p38 MAPK was
increased after H 2 O 2 treatment and lasted for 24 hours, while phosphorylation of
ERK and Akt were unaltered under the same treatment. The enhanced activity of
voltage sensitive Ca 2+ channel and bradykinin-IP 3 signaling pathway and
enlargeed Ca 2+ pool caused by H 2 O 2 were reversed by simultaneously
incorporation of SB203580, while remained the same by PD98059 and
LY294002, respectively. Taken together our result show that in NG108-15 cells,
low dose of H 2 O 2 promotes differentiation. The effect were through the
activation of p38 MAPK.

正文目錄
正文目錄 I
圖文目錄 II
縮寫表 III
摘要 V
Abstract VI
第一章 緒論 1
第二章 實驗材料與方法 4
第一節、實驗材料 4
壹、細胞材料 4
貳、藥品試劑 4
參、實驗儀器 6
第二節、實驗方法 7
壹、NG108-15 細胞培養 7
貳、細胞內鈣離子濃度測定 7
參、細胞型態 8
肆、細胞凋亡測定 9
伍、細胞自噬測定 9
陸、西方墨點法(Western) 10
第三章 實驗結果 12
第一節 NG108-15細胞生長之影響 12
壹、細胞型態之觀察 12
貳、H2O2對細胞生長的影響 13
第二節 NG108-15細胞分化特徵之觀察 14
壹、神經突之量化 14
貳、VSCC活性 14
第三節 H2O2對NG108-15細胞分化機制探討 16
壹、H2O2刺激NG108-15細胞訊息傳遞路徑之探討 16
貳、ERK、Akt、p38MAPK與細胞分化之關聯性 18
第四章 討論 19
第五章 結論 21
第六章 參考文獻 31


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