端粒酶活化被認為是形成腫瘤的特徵之一，藉由維持端粒長度來避免腫瘤老化。此外，惡性腫瘤中癌症幹細胞對於化學藥物治療、放射線治療具有抗性，被認為是造成腫瘤復發之主因。在先前研究已發現小分子藥物 DH-001 在人類惡性腦膠質瘤細胞中可明顯抑制端粒酶的表現，進而使惡性腦膠質瘤細胞走向老化途徑，因此本研究之目的為利用小分子藥物 DH-001 探討惡性腦膠質瘤幹細胞與惡性腦膠質瘤細胞調控老化機制之差異。DH-001 具有抑制惡性腦膠質瘤幹細胞的生長能力，然其端粒酶表現量無明顯受到抑制。根據 β-半乳糖染色結果，發現加入 DH-001 後，相較於惡性腦膠質瘤細胞，惡性腦膠質瘤幹細胞未出現老化之現象。因此為瞭解 DH-001 無法抑制惡性腦膠質瘤幹細胞老化的機制，本研究進一步探討 DH-001 與端粒酶反轉錄酶啟動子上轉錄因子 GA-binding protein (GABP) 之間的作用關係。實驗結果發現惡性腦膠質瘤細胞及惡性腦膠質瘤幹細胞皆有表現突變熱點。加入 DH-001 後，惡性腦膠質瘤細胞的 GABPβ 表現量受到 DH-001 抑制，而惡性腦膠質瘤幹細胞則否。經由 shRNA 轉染，可發現 GABPβ表現之抑制，可提升 DH-001 對惡性腦膠質瘤幹細胞之毒殺效力、提升老化相關的基因如 p16 與 p27 表現量，以及降低端粒酶之表現量。綜合以上研究結果，惡性腦膠質瘤細胞及惡性腦膠質瘤幹細胞對於基因 GABPβ 調控端粒酶路徑的不同，說明 GABPβ之調控在惡性腦膠質瘤幹細胞之抗老化可能扮演關鍵角色。

The abnormal activation of telomerase, which prevents senescence effect of tumor by maintaining the telomere length, is one of the characteristics for tumor formation. In addition, the resistance of cancer stem cells on chemotherapy and radiotherapy is regarded as the main cause of tumor recurrence. The DH-001 was previously found to undergo senescence pathway of glioblastoma cells via inhibiting the expression of telomerase, therefore DH-001 was used to investigate the difference of senescence modulating effect between glioblastoma stem cells (GSCs) and glioblastoma cells (non-GSCs) in this study. The results indicated that the progressing ability of GCSs was inhibited by DH-001, whereas their expression of telomerase was not. In comparison with non-GSCs, the GSCs did not undergo senescence after DH-001 treatment according to the results of β-galactose staining. To further determine the mechanism for which DH-001 was unable to inhibit the senescence of GSCs, the relationship between DH-001 and the transcription factor GABP of TERT promoter was analyzed. The results revealed that the hot spot of mutation on TERT promotor could be detected in both GSCs and non-GSCs. The treatment of DH-001 inhibited the expression of GABPβ on non-GSCs, but not on GSCs. Furthermore, the inhibition of GABPβ by shRNA could enhance the cytotoxic effect of DH-001, upregulate the expression of senescence genes such as p16 and p27, as well as downregulate the expression of telomerase in GSCs. Taken together, the difference of GABPβ on regulating telomerase between GSCs and non-GSCs suggests that GABPβ serves as a key role in anti-senescence mechanism for GSCs.

中文摘要 Ⅰ
abstract Ⅲ
目錄 1
一、 研究動機與研究目的 5
二、 研究背景介紹 7
2.1 人類惡性腦膠質瘤 7
2.1.1 腦瘤的分類及進程 7
2.1.2 腦瘤的診斷 9
2.1.3 腦瘤的治療 9
2.2 癌症幹細胞 10
2.2.1惡性腦瘤CD133+ 11
2.4 端粒與端粒酶 12
2.5 TERT 啟動子突變 13
2.6 GA-binding protein (GABP) 轉錄因子之功能 14
2.7 GABP與TERT啟動子突變之關係 14
2.8 DH-001 15
三、 材料與方法 17
3.1 實驗流程設計 17
3.2 細胞來源與培養 17
3.3 MTT assay 18
3.4 細胞total RNA之抽取 19
3.5 反轉錄聚合酶連鎖反應 (reverse transcription-PCR, RT-PCR) 19
3.6 即時定量聚合酶連鎖反應 20
3.7全細胞之蛋白質抽取及蛋白質濃度測定 21
3.8西方墨點分析法 22
3.9端粒酶活性分析 (TRAP assay) 23
3.10端粒酶活性酵素連結免疫吸附分析 (ELISA) 23
3.11端粒長度分析 24
3.12細胞衰老β-半乳糖染色 25
3.13 TERT啟動子突變分析 25
3.14細胞基因靜默 (gene silencing) 26
3.15統計分析 27
四、 結果 29
五、 實驗結果附圖 35
六、 討論 49
七、 結論 57
八、 參考文獻 59

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