臺灣博碩士論文加值系統

多型性神經膠質母細胞瘤(Glioblastoma multiforme，GBM)為成人中最常見且惡性的致命性原生腦癌，被世界衛生組織(WHO)歸列為第四級神經膠質瘤。多型性神經膠質母細胞瘤(GBM)具有高度異質性與侵略性，並對於傳統的化學及放射治療有抗藥性，因此多型性神經膠質母細胞瘤(GBM)之病患即使經過治療，仍有極高機率會復發，故其平均存活率不超過16個月。而此現象可能與GBM之癌幹細胞密切相關，因此針對癌幹細胞做為治療GBM的策略為一重要之目標及方向。中草藥在中國已被廣泛使用數千年，現今則有研究指稱它具有治療某些癌症疾病的潛能。因此，我們以腦癌細胞株U251-MG及U373-MG及其衍生之tumorsphere建立篩藥平台，以篩選可抑制GBM生長之有效中草藥萃取物。我們從200種中草藥酒萃物中篩選出兩種中草藥萃取物可抑制U251及U373 tumorsphere之存活，但對於正常的老鼠星狀膠細胞毒性較低。此兩種萃取物可能藉由調控Akt或ERK之訊息傳遞路徑，並影響細胞週期而達到抑制細胞生長，另外它們可降低幹細胞標記—CD133或Sox2之表現。此外，我們發現化合物BS可能是IT229中有效對抗GBM幹細胞之有效成分，其可有效降低U373-tumorsphere之存活。綜上所述，此兩種中草藥萃取物可能為有潛能治療GBM之藥物，並期許其未來可應用於臨床治療上。

Glioblastoma multiforme (GBM), classified as the grade IV astrocytoma, is the most common, malignant and fetal primary brain cancer in adult. GBM possess the characters of heterogeneity, invasiveness and resistance to traditional therapies. The GBM patients often experienced tumor relapse after aggressive treatment, therefore the median survival of the patients is about 14.6 month. Recent studies indicated that the glioblastoma stem cells (GSCs) may play the critical role in tumor relapse, thus targeting GSCs to against GBM is one of the potential therapeutic strategies for GBM. Chinese herbal medicine has been applied widely in Chinese society for thousands of year, and it is relatively mild and has less side-effect compared to the modern western medicine. Therefore, we used human glioblastoma cell lines and their derived tumorspheres to set up a platform for screening effective herbal extracts. In this study, we have screening 200 alcoholic herbal extracts and found two of them which inhibit cell viability of U251 and U373 tumorspheres but with low toxicity to normal mouse astrocytes. Furthermore, they reduce the ability of tumorsphere formation and cell growth by inducing cell cycle arrest. In addition, the two herbal extracts inhibit Akt or ERK pathway and down-regulate the expression of stem cell marker-CD133 or Sox2. Moreover, we found that compound –BS may be the bioactive component in IT229 and it also inhibit cell viability of U373 tumorspheres. In conclusion, the two herbal extracts may have therapeutic potential in GBM treatment through targeting glioblastoma stem cells.

目錄 --------------------------------------- i
縮寫表 ------------------------------------- iii
中文摘要 ---------------------------------------- 1
英文摘要 ---------------------------------------- 3
1. 緒論 ------------------------------------- 5
1.1 腦癌(Brain cancer) --------------------------- 5
1.2 多型性神經膠質母細胞瘤(GBM) -------------------- 6
1.3 中草藥 (Chinese herbal medicine) ------------- 8
2. 研究方法與材料 ---------------------------------- 10
2.1 中草藥萃取物 ---------------------------------- 10
2.2 細胞培養 --------------------------------- 10
2.3 MTT Assay ---------------------------------11
2.4 WST-1 Assay --------------------------------- 12
2.5 西方墨點法 ---------------------------------- 13
2.6 群落形成分析 ---------------------------------- 14
2.7 CD133+細胞分離 ------------------------------- 15
2.8 細胞週期分析 ------------------------------ 16
2.9 AnnexinV及PI 雙重染色--------------------------- 17
2.10 統計分析 -------------------------------------- 17

3. 結果
3.1 植物酒萃物影響GBM及其幹細胞生長之探討
3.1.1 植物酒萃物有效抑制U251-MG細胞生長-------------------18
3.1.2 植物酒萃物有效抑制U251及U373-tumorsphere之生長-----19
3.1.3 植物酒萃物降低U251-tumorsphere形成Colony之能力-----21
3.1.4 植物酒萃物有效抑制GBM之幹細胞生長-------------------22
3.1.5 植物酒萃物對於小鼠神經膠細胞及神經幹細胞之毒性測試---23
3.2 植物酒萃物抑制GBM幹細胞生長之機轉探討
3.2.1 植物酒萃物藉由影響細胞週期抑制GBM之生長--------------24
3.2.2 植物酒萃物減少GBM幹細胞標記蛋白質表現，並降低
ERK或Akt之活化 -----------------------------------------27
3.3 植物酒萃物之有效活性成分抑制GBM幹細胞生長之分析
3.3.1 BS抑制U373-tumorsphere細胞生長------------------- 28
3.3.2 BS對於小鼠神經膠細胞及神經幹細胞之細胞毒性測試------ 29
3.3.3 BS誘導U373-tumorsphere發生細胞凋亡------------------ 29
3.3.4 BS減少幹細胞之相關蛋白質表現，並降低Akt之活化------ 30
4. 討論 ---------------------------------------31
5. 參考文獻 -------------------------------------- 37
6. 圖表 -------------------------------------- 43

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