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研究生:蔡昇峰
研究生(外文):Sheng-Fong Tsai
論文名稱:異柴胡內酯提升內質網壓力相關蛋白DDIT3調控 NAG-1 表現促使多形性腦膠質瘤細胞凋亡
論文名稱(外文):Endoplasmic Reticulum Stress Related Protein DDIT3 modulated NAG-1 Revealed Isochaihulactone-triggered Apoptotic Pathway in Human Glioblastoma Multiform Cells
指導教授:蘇鴻麟蘇鴻麟引用關係
口試委員:邱紹志韓鴻志
口試日期:2014-07-22
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:39
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外文關鍵詞:no
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多形性神經膠質母細胞瘤( Glioblastoma multiform , GBM )因其侵犯能力強、生長快速與預後不良被譽為最惡性腦瘤類型。目前現有臨床治療以手術切除大部分病灶輔以放射治療及化學治療僅能延長 6 個月壽命且復發率極高,急需發展出一種能夠有效抑制 GBM 生長新型藥物。先前研究指出異柴胡內酯 ( K8 ) 在攝護腺癌與肺癌中透過活化腫瘤細胞中 NAG-1 基因表現使其凋亡,是一種非常有潛力的小分子藥物,但 K8 抑制癌細胞的作用機制尚未完全了解。本次實驗發現在 K8 也會活化 GBM 細胞中 NAG-1 表現,使惡性腦瘤細胞周期 sub-G1 時期比例上升,也
活化細胞凋亡相關蛋白 Caspase3、Caspase9。為了探討異柴胡內酯是透過甚麼路徑造成 GBM 細胞凋亡,我們進一步使用微陣列分析掃描 K8 處理 GBM 細胞 24 小時後基因表現變化,發現 K8 會活化 GBM 細胞中內質網壓力相關蛋白 DDIT3、ATF3及 NAG-1 等基因表現,其中 DDIT3 活化 5.73 倍,NAG-1 4.38 倍。DDIT3 在細胞中會因為內質網壓力 ( Endoplasmic reticulum stress , ER stress ) 過載而進行細胞凋
亡時表現。透過反轉錄聚合酶鏈鎖反應與西方墨點法確認 GBM 細胞在 K8 的處理下 NAG-1 及 DDIT3 表現量隨著處理濃度升高而提高。為了更進一步探討 K8 是否是透過活化 NAG-1 與 DDIT3 造成 GBM 細胞凋亡。我們利用核醣核酸干擾 ( RNA interference ) 技術 將 NAG-1 與 DDIT3 的小片段干擾 RNA ( short interference RNA,,
siRNA ) 傳入 GBM 細胞中以降低 K8 誘發的 NAG-1 與 DDIT3 基因表現,發現在RNA 干擾的情況下會減少 K8 對 GBM 細胞的毒殺效果,細胞活性回復了 23.4 %。為了確例 DDIT3 可能為調控 NAG-1 之假說,發現在抑制 DDIT3 表現下 NAG-1 表現量會因 DDIT3 被抑制而表現量下降;在抑制 NAG-1 表現的情況下異柴胡內酯仍會誘發 DDIT3 表現,根據此結果推測 DDIT3 可能為 NAG-1 的調控基因。綜合以上實驗結果說明異柴胡內酯會透過活化 DDIT3 調控 NAG-1 表現使得惡性腦膠母細胞瘤凋亡。此發現可能為治療惡性腦瘤膠著現況中提供一個新的標靶基因。


Glioblastoma multiforme ( GBM ) is the most malignant brain tumor characterized by aggressive and invasive behavior, and a poor prognosis. There is a critical need to identify novel drugs targeting GBM and to develop more effective therapeutic strategies. Previous studies, showed that isochaihulactone ( K8 ) would cause cell apoptosis in lung
cancer and prostate cancer by activating the nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression. K8 which showed dramatic anti-tumor ability is a potential small molecular drug to be investigated. In this study, we found that K8 also caused GBM cell apoptosis and activated NAG-1 expression in does-dependent treatment. However, the underlying molecular mechanisms of K8 causing cancer cell line apoptosis remain not clear. We used microarray screening to find that K8 induced DDIT3, ATF3 and NAG-1 expression in GBM cells. Here we showed that DDIT3, a endoplasmic reticulum stress related protein induced by K8 treatment. We hypothesized that DDIT3 modulate NAG-1 expression causing cell death. First we used reverse transcription polymerase chain reaction ( RT-PCR ) and Western Blot to confirm K8 induced NAG- 1,DDIT3 gene expression in GBM cell, whereas RNAi-mediated knockdown of DDIT3 and NAG-1 reduce K8 cell toxicity. Importantly, when we used siRNA to knockdown DDIT3 expression , the NAG1 mRNA expression level depression as well. On the contrary, we knockdown NAG-1 and DDIT3 induced by K8. In conclusion, our result supported the hypothesis that K8 would cause GBM cell apoptosis via active DDIT3 modulating NAG1 tumor suppressor pathway.


誌謝詞 .................................................. i

中文摘要 ........................................ ii

Abstract ...................................... iii

第一章 前言 ................................... 1
1-1 腦瘤 ....................................................... 1
1-2 腦瘤的分類....................................... 1
1-3 人類惡性腦膠質瘤 ............................. 2
1-4 人類多形性腦膠母細胞瘤可能形成原因 ........... 3
1-5 中草藥抗腫瘤能力 ............................ 4
1-6 南柴胡抗癌成分篩選 .......................... 4
1-7 非類固醇抗消炎活化基因 (Non-steroid anti-inflammation active gene-1 , NAG-1 )
與惡性腫瘤 ...5
1-8 內質網壓力 ( Endoplasmic reticulum stress ) .......... 6

第二章 材料與方法........................................ 8
2-1 細胞培養.............................................. 8
2-2 細胞活性測試.......................................... 8
2-3 細胞週期分析............................................. 8
2-4 全細胞核醣核酸抽取 ................................... 9
2-5 Trizol 法細胞核醣核酸抽取 ............................... 9
2-6 微陣列晶片分析 ...................................... 10
2-7 反轉錄聚合酶連鎖反應 ................................ 11
2-8 全細胞蛋白質抽取及蛋白濃度測定 ...................... 11
2-9 西方墨點法........................................... 12
2-10 細胞轉染.......................................... 13
2-11 統計分析......................................... 14
第三章結果 ............................................. 15
3-1 異柴胡內酯抑制人類惡性腦膠母細胞瘤 8401 細胞活性 ...... 15
3-2 異柴胡內酯會造成人類惡性腦瘤細胞凋亡而非細胞週期停滯 ........15
3-3 微陣列晶片分析 ................................................ 16
3-4 在異柴胡內酯處理下 GBM 8401 中的 NAG-1 RNA 與 Preotein 表現上升 .... 16
3-5 異柴胡內酯透過誘發 NAG-1 使得 GBM8401 細胞活性下降 .............. 17
3-6 在異柴胡內酯處理下 GBM 8401 中的 DDIT3 RNA 與 Protein 表現上升..... 17
3-7 異柴胡內酯透過誘發 DDIT3 使得 GBM8401 細胞活性下降 .............. 18
3-8 異柴胡內酯提升 DDIT3 表現調控下游 NAG-1 使得 GBM8401 細胞凋亡..... 18

第四章 討論 ................................. 20

第五章 圖與表...............................25
圖一、異柴胡內酯分子結構。 ..................................... 25
圖二、異柴胡內酯抑制惡性腦膠質瘤細胞 8401 活性。(A) ................................................ 26
圖三、惡性腦膠質瘤細胞 8401 在異柴胡內酯 60μM 處理下細胞週期變化。.................. 27
圖四、異柴胡內酯誘發惡性腦膠質瘤 8401 細胞凋亡相關蛋白之表現。 ......................... 28
圖五、異柴胡內酯活化人類 GBM 8401 表現 NAG-1。 ........................................................ 29
圖六、降低 NAG-1 表現可回復異柴胡內酯抑制人類惡性腦瘤細胞之活性。 ................... 30
圖七、異柴胡內酯活化人類 GBM 8401 表現 DDIT3。 ......................................................... 31
圖八、降低 DDIT3 表現可回復異柴胡內酯抑制人類惡性腦瘤細胞之活性。 ................... 32
圖九、降低 DDIT3 mRNA 表現量也會抑制下游 NAG-1 mRNA 表現量。 ............................ 33
表一、本實驗使用核酸引子序列及產物長度 ............................................. 35
表二、GBM8401 經異柴胡內酯半致死劑量處理 24 小時後被誘導表現基因。 ................ 35

第六章 參考文獻...................................... 36


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