臺灣博碩士論文加值系統

Chronic myeloid leukemia （CML）是一種由 Bcr-Abl 融合基因引起的骨髓增殖性疾病 ，其中 T315I 突變使 CML 細胞對 imatinib 產生耐藥性 。第三代酪氨酸激酶抑製劑 ponatinib 雖然可以抑制 Bcr-Abl 激 酶 ，但也存在嚴重的副作用 。Warburg effect ，即有氧糖酵解，是腫瘤細胞獲取能量的重要來源 。先 前的研究證明 ，抑制 Warburg effect 可能是一種有前途的癌症治療策略 。本研究旨在探討 rigosertib 和 Asiatic Acid(AA)對 T315I 突變 CML 細胞 的影響及機制 。 Rigosertib 是一種非 ATP 競爭性多靶向 抑製劑 ，可抑制 CML 細胞增殖 。AA 是一種天然小分子藥物 ， 可誘導實體瘤細胞凋亡 。我們首先發現 AA 在 48 小時處理後抑制 K562、BaF3/p210 和 BaF3/T315I 細胞的增殖並誘導細胞凋亡 。 Rigosertib 和 AA 具有誘導 K562、BaF3/p210 和 BaF3/T315I 細胞凋亡的能力 。AA 在處理 48 後能 夠增加 c-caspase3/PARP 蛋白量表達 。Rigosertib 和 AA 在處理 48 小時後能夠下調 K562、 BaF3/p210 和 BaF3/T315I 細胞 HK1、HK2、HIF1α、PDK1 和 Glut1 Warburg effect 相關基因 mRNA 的表達 。Rigosertib 能夠誘導 K562、BaF3/p210 和 BaF3/T315I 細胞凋亡，並下調 HK2，PDK1 蛋白 表達與增加細胞週期的 G2/M 期停滯 。這些結果表明 ，rigosertib 和 AA 可通過抑制具有 T315I 突變 的 CML 細胞的 Warburg 效應來誘導生長抑制和細胞凋亡 。

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by the Bcr-Abl fusion gene, in which the T315I mutation makes CML cells resistant to imatinib. Although the third-generation tyrosine kinase inhibitor ponatinib can inhibit Bcr-Abl kinase, it also has serious side effects. Warburg effect, known as aerobic glycolysis, is an important source of energy acquisition for tumor cells. Previous studies showed that inhibition of Warburg effect may be a promising strategy for cancer therapy. The aim of this study was to explore the influence and mechanism of rigosertib and asiatic acid (AA) on Warburg effect in CML cells with T315I mutation. Rigosertib is a non-ATP competitive multi-targeting inhibitor. It can induce growth inhibition and apoptosis in CML cells with Bcr-Abl T315I mutation. AA is a natural small-molecule drug that also can induce growth inhibition and apoptosis in solid tumor cells. We first found that AA inhibited the proliferation and induced apoptosis in K562, BaF3/p210, and BaF3/T315I cells after 48h treatment. Rigosertib and AA were able to down regulate the Warburg effect related genes HK1, HK2, HIF1α, PDK1, and Glut1 mRNA expression after 48h treatment. Rigosertib induced apoptosis in K562, BaF3/p210, and BaF3/T315I cells and down regulated HK2 and PDK1 proteins level and increased cell cycle G2/M arrest. These results suggested that rigosertib and AA induce growth inhibition and apoptosis by inhibiting Warburg effect in CML cells with T315I mutation.

目錄Ⅰ
表目錄Ⅲ
中文摘要Ⅳ
AbstractⅤ
第一章：緒論1
1.1 Chronic myelocytic leukemia(CML)1
1.2 Warburg effect4
1.3 Warburg effect相關調控蛋白4
1.4 新型小分子藥物Rigosertib與Asiatic acid5
研究目的7
第二章：材料與方法8
2.1 實驗藥物配製8
2.2 細胞培養8
2.3 細胞數量計算8
2.4 細胞存活率分析（MTT assay）9
2.5 流式細胞儀分析（Flow cytometry assay）9
2.6 蛋白質萃取與Western blot10
2.7 RNA萃取11
2.8 RT-PCR12
2.9 統計方法12
第三章：實驗結果14
3.1 Asiatic acid抑制CML細胞存活率14
3.2 Rigosertib與AA誘導CML細胞凋亡14
3.3 AA增加c-PARP與c-caspase3蛋白表達水平15
3.4 Rigosertib與AA下調CML細胞中Warbrug effect相關mRNA表達15
3.5 Rigosertib下調CML細胞中Warbrug effect相關蛋白水平15
3.6 Rigosertib使CML細胞週期停滯在G2/M期16
第四章：討論17
第五章：圖表19
圖1. AA抑制K562， BaF3/p210，BaF3/T315I細胞存活率19
圖2. AA與Rigosertib誘導K562， BaF3/p210，BaF3/T315I細胞凋亡20
圖3. AA通過c-caspase3/PARP路徑誘導CML細胞凋亡21
圖4. Rigosertib與AA下調CML細胞中Warbrug effect相關mRNA表達22
圖5. Rigosertib下調CML細胞中Warbrug effect相關蛋白水平23
圖6. Rigosertib使CML細胞週期停滯在G2/M期：24
參考文獻：25
表目錄
實驗使用引子12

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