多型性膠質母細胞瘤(Glioblastoma multiforme)為最惡性的腦瘤，具有高度的致其周邊組織壞死、細胞遷移與新生血管的特性。經存活率統計，在傳統的外科手術及放射治療與化學治療，平均存活時間仍不超過15個月，是目前極為棘手且治癒甚差的極惡性腫瘤。其中血管新生是腫瘤發展必經的途徑之一，抗血管新生亦為一重要癌症藥物發展之策略；血管新生過程中，血管內皮生長因子(Vascular endothelial growth factor A, VEGF-A)為其一重要的促血管新生因子，癌細胞透過釋放VEGF使周圍血管內皮細胞的VEGF receptor (VEGFR)接受VEGF，進而促進血管內皮細胞增生，產生新生血管。自從發現VEGF在血管新生中的角色後，便成了後續將其作為抗血管新生標靶治療的重要依據。
本研究所使用之海洋化合物PON-1621來自海綿Jaspis stellifera之萃取物，前人研究其具有抗癌活性之現象，但未有文獻釐清其機制。本研究結果顯示，此化合物透過抑制AKT與STAT3磷酸化，減少girdin與VEGF的mRNA表現，進而達到抑制血管新生與細胞遷移的功效，以此結果推測，PON-1621或許可成為具有潛力的抗腫瘤血管新生用藥。

As the most aggressive brain cancer type, glioblastoma multiforme (GBM) has features of high level of tissue necrosis, cell migration, and angiogenesis. The survival ratio of GBM is less than 15 months, although treat with traditional surgical, radiotherapy, and chemotherapy. It’s an extremely severe malignant tumor, and can’t be cured so far. In GBM, angiogenesis plays an important role in tumor progression. Therefore, anti-angiogenesis could be one of effect way to stop or slow the growth of GBM. VEGF-A is an important ligand of neovascularization. The tumor cell induces angiogenesis by paracrine of VEGF-A, stimulates VEGFR expression on endothelia cells nearby then. In order to the importance of VEGF-A on tumor angiogenesis, it became a key target of anti-angiogenesis therapy.
A marine-derived bioactive compound, PON-1621, which was extracted from marine sponge, Jaspis stellifera, exibits some uncleared anti-cancer bioactivities. In this research, PON-1621 down-regulates mRNA expression of girdin and VEGF, which are important for migration and angiogenesis by AKT/mTOR/S6 and STAT3 signaling pathway blocking. Based on these results, PON-1621 may be a potential agent on tumor therapy for it’s anti-angiogenesis ability.

論文審定書 i
誌謝 ii
摘要 iii
Abstract iv
目錄 v
圖次 ix
表次 xi
第壹章、前言 1
第一節、惡性腫瘤之特色 1
第二節、中樞神經系統腫瘤 (Central nervous system tumor) 2
一、多型性膠質母細胞瘤 (Glioblastoma multiforme) 5
二、目前惡性腦瘤之臨床治療策略 6
三、FDA通過之惡性腦瘤使用藥物 7
第三節、惡性腫瘤之訊息傳遞路徑 9
一、AKT/mTOR/S6訊息傳遞路徑 (AKT/mTOR/S6 signaling pathway) 9
二、STAT3訊息傳遞路徑 (STAT3 signaling pathway) 13
第四節、惡性腫瘤轉移之機制 14
一、癌症轉移 (metastasis) 14
二、血管新生 (angiogenesis) 16
第五節、現行惡性腫瘤標靶藥物 20
一、現有之惡性腫瘤標靶藥物 20
二、抑制AKT/mTOR/S6訊息傳遞路徑之藥物 21
第六節、海洋天然藥物 24
一、已上市之海洋抗癌藥物 24
第七節、研究動機與目的 27
第貳章、實驗材料與方法 28
第一節、細胞培養 28
一、細胞株 28
二、細胞繼代培養 28
三、細胞計數 28
第二節、細胞存活率測試 29
第三節、離體細胞Caspase-3活性表現測試 (Caspase-3 activity assay) 29
第四節、離體細胞之血管生成作用測試 (Tube formation assay) 29
第五節、離體細胞之遷移作用測試 30
一、Wound healing migration assay 30
二、Invasion assay 30
第六節、PON-1621對腦瘤細胞蛋白與基因表現之影響 31
一、蛋白表現量測定 (西方點墨法) 31
二、基因表現量測定(即時聚合酶連鎖反應) 34
第七節、VEGF胞外分泌量測定 35
第八節、PON-1621對p-Girdin與F-actin間交互作用之影響 36
第九節、斑馬魚血管新生模式 37
一、斑馬魚種魚Tg (fli1:EGFP)飼養 37
二、斑馬魚胚胎取得 37
三、藥物濃度篩選與實驗仔魚飼養 37
四、斑馬魚體節間血管新生 37
第十節、斑馬魚mRNA分析 38
一、斑馬魚仔魚mRNA萃取 38
第十一節、小鼠血管新生模式 39
一、小鼠BALB/c飼養 39
二、Matrigel Plug Assay 39
三、Drabkin’s Reagent血紅素定量 39
第十二節、實驗數據分析 40
第參章、實驗結果 41
第一節、PON-1621對膠質母細胞瘤細胞株型態與存活之影響 41
一、細胞型態變化 41
二、細胞存活率 41
三、PON-1621對Caspase-3活化誘導的膠質母細胞瘤細胞凋亡之影響 42
第二節、離體細胞之血管生成作用測試 (Tube formation assay) 42
一、PON-1621對人類臍靜脈內皮細胞HUVEC存活之影響 42
二、PON-1621對HUVEC細胞血管生成作用之影響 42
第三節、PON-1621對膠質母細胞瘤細胞遷移作用之影響 43
一、PON-1621對膠質母細胞瘤細胞入侵作用之影響 (Invasion assay) 43
二、PON-1621對GBM8401細胞遷移之影響 (Wound Healing assay) 43
第四節、PON-1621對膠質母細胞瘤血管新生相關路徑之影響 43
一、PON-1621對膠質母細胞瘤細胞AKT活化之影響 44
二、PON-1621對膠質母細胞瘤細胞mTOR活化之影響 44
三、PON-1621對膠質母細胞瘤細胞S6活化之影響 44
四、PON-1621對膠質母細胞瘤細胞STAT3活化之影響 44
五、PON-1621對膠質母細胞瘤細胞HIF-1α mRNA與蛋白表現之影響 45
六、PON-1621對膠質細胞瘤細胞VEGF-A mRNA與蛋白表現之影響 45
七、PON-1621對膠質母細胞瘤細胞VEGF分泌之影響 45
第五節、PON-1621對膠質母細胞瘤細胞遷移相關蛋白之影響 46
一、PON-1621對膠質母細胞瘤細胞EMT相關蛋白表現之影響 46
二、PON-1621對膠質母細胞瘤細胞Girdin mRNA表現之影響 46
三、PON-1621對膠質母細胞瘤p-Girdin活化之影響 46
四、PON-1621對膠質母細胞瘤p-Girdin與F-actin間交互作用之影響 47
第六節、PON-1621對斑馬魚仔魚血管新生之影響 47
一、PON-1621對斑馬魚仔魚存活率之影響 47
二、PON-1621對血管螢光斑馬魚胚胎之體節間血管形成的影響 48
三、PON-1621對Tg (fli1:EGFP)斑馬魚VEGF-A mRNA表現之影響 48
第七節、PON-1621對BALB/c小鼠血管新生之影響 48
第肆章、討論 80
第一節、AKT/mTOR/S6訊息傳遞路徑對癌細胞生長之重要性 80
第二節、PON-1621抑制AKT/mTOR/S6及STAT3訊息傳遞路徑所促進膠質母細胞瘤促進血管新生之機制探討 81
第三節、Girdin在膠質母細胞瘤細胞之遷移現象中之新興角色 83
第四節、PON-1621在in vitro與in vivo實驗抑制血管新生現象 85
第五節、克服PON-1621藥物毒性之策略 86
第六節、腦瘤血腦障壁之突破 86
第七節、總結 86
第伍章、未來展望 90
第陸章、參考文獻 91

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