Imatinib (Gleevec)是治療慢性骨髓性白血病臨床的第一線標靶藥物，但疾病復發頻繁且容易伴隨著抗藥性的發生。癌症幹細胞學說是近年癌症生物學非常重要之進展，此理論不但提供慢性骨髓性白血病生成之可能原因，更解釋慢性血癌產生抗藥性導致難以治癒之可能機轉。因此，在慢性骨髓性白血病的治療上如何標靶及清除癌症幹細胞是一項很重要之研究議題。我們利用GEO資料庫系統分析發現組蛋白去乙醯?＆a族（HDAC）在CML患者之高度表現與Imatinib抗藥性具有相關性。因此本研究之目的主要探討Imatinib抗藥性與慢性血癌幹細胞生成之關聯及合併HDAC抑製劑來治療Imatinib抗藥性之可能性。首先，我們以K562慢性血癌細胞為模式，利用FACSaria分選並鑑定CD34(+) / CD38(-)細胞具有高度Oct4 ,CD133, β-catenin和Sox2等癌幹細胞基因表現。此外，我們還證實CD34(+) / CD38(-)細胞亦有高度表現HDAC家族和抗藥性基因，推測與Imatinib抗藥性有相關。有趣的是，我們也發現HDAC抑製劑：SAHA可增強Imatinib抑制CD34(+) / CD38(-)細胞生長之能力並且可以藉由調控細胞週期的蛋白（CDK , P18 , P21和P27）和細胞凋亡蛋白（BCL - 2和BAX）的表現增加來誘導CD34(+) / CD38(-)細胞進行凋亡。從機制分析，我們認為合併SAHA及Imatinib治療可以降低細胞內HDAC 4 , 5和cyclin D3的表現，進而提升細胞對Imatinib敏感性增加。藉此研究，我們證實了CD34(+) / CD38(-)細胞具高度表現HDAC基因能力，這種高表達與Imatinib抗藥性有關。加入HDAC抑製劑：SAHA 使得對Imatinib抗藥性的癌症幹細胞對淤Imatinib再度敏感化。這種藥物之組合有可能被用於未來臨床的使用，以減少對Imatinib的抗藥性和CML復發。

Imatinib (Gleevec) is the clinical first line agent for treating chronic myeloid leukemia (CML), but disease recurrence are frequent and accompanied with resistance. One of the major contributors for imatinib-resistance is leukemia-initiating cells (LICs). Thus, targeting and eliminating LICs represents an important task for treating CML. Through public GEO database analysis, we have identified that a high expression level of histone deacetylases (HDAC) is associated with imatinib resistance in CML patients. In this study, we aimed to investigate the relevance of Imatinib -resistant cells and CD34 + cells in the generation of CML stem cells and the usage of HDAC inhibitor for possible combination therapy. First, we isolated and identified CD34(+)/CD38(-) cells with high expression of Oct4, CD133, β-catenin, and Sox2. Furthermore, we also demonstrated that high expression of HDAC and drug resistant gene were found in CD34(+)/CD38(-) LICs and associated with their resistance to imatinib. Importantly, the usage of HDAC inhibitor, SAHA, in combination with Imatinib sensitized CD34(+)/CD38(-) LICs and induced apoptosis, shown by increased expression of cell cycle (CDK2, P18, P21, and P27) and apoptotic proteins (BCL-2 and BAX). Mechanistically, the combined treatment reduced HDAC 4, 5 and cyclin D3 protein expression, which led to increased sensitivity toward SAHA and Imatinib. In conclusion, we demonstrated that LICs (CD34+/CD38-) express a high level of HDAC and this high expression was associated with imatinib resistance. Adding HDAC inhibitor, SAHA, re-sensitized imatinib-resistant LICs to imatinib. This combination could potentially be employed for future clinical settings to reduce imatinib resistance and CML recurrence.

目錄
致謝 - 1 -
目錄 - 3 -
摘要 - 6 -
Abstract - 7 -
第一章 緒論(Introduction) - 8 -
(一)前言 - 9 -
(二) 白血病(Leukemia)的介紹 - 9 -
(三) 慢性骨髓性白血病(chronic myeloid leukemia；CML)的介紹 - 11 -
(四) BCR-ABL致癌蛋白質 - 14 -
(五) 白血病幹細胞的特性 - 14 -
(六) Imatinib: indication of CML - 15 -
(七) CML復發的原因 - 16 -
(八) Histone deacetylases, HDACs - 17 -
(九) Histone deacetylases, HDACs的分類 - 18 -
(十) HDAC inhibitors, HDACi的功能 - 19 -
(十一) HDAC inhibitor(Histone deacetylase inhibitor)--- Vorinostat(SAHA) - 19 -
第二章 實驗動機目的與設計 - 21 -
第三章 實驗材料與方法(Materials and Methods) - 23 -
(一) 選用細胞 - 24 -
(二) 藥品試劑與常用溶液 - 25 -
(三) 常用儀器 - 29 -
(四) 實驗方法 - 30 -
(1) 細胞解凍、繼代、冷凍保存 - 30 -
(2) 細胞存活率測定(trypan blue exclusion) - 31 -
(3) RNA extraction & RNA濃度測定 - 31 -
(4) 反轉錄—聚合酶連鎖反應 (RT-PCR) - 32 -
(5)即時定量聚合酶連鎖反應(Real-time PCR) - 33 -
(6) 蛋白質萃取 - 33 -
(7) 蛋白質測定 - 33 -
(8) 西方墨點法(Western Blotting Assay) - 34 -
(9) Cell cycle analysis - 35 -
第四章 實驗結果(Results) - 36 -
(一) 分離與觀察白血病幹細胞的幹性基因表現 - 37 -
(二)白血病幹細胞相關蛋白質的幹性基因表現 - 37 -
(三) 白血病幹細胞在HDAC上的蛋白表現 - 38 -
(四) Imatinib對白血病幹細胞的影響 - 39 -
(五) 使用HDAC inhibitor-SAHA與IM分別治療對CD34+/CD38-白血病細胞的影響 - 39 -
(六) 使用HDAC inhibitor-SAHA與IM合併治療對CD34+CD38-白血病幹細胞的影響 - 40 -
(七) HDAC inhibitor-SAHA與IM合併治療後對白血病幹細胞的蛋白表現 - 40 -
(八) 白血病幹細胞經HDAC inhibitor-SAHA與IM合併治療後其細胞生長週期的表現 - 41 -
第五章 實驗討論 - 43 -
第六章 結論 - 49 -
第七章 圖表 - 51 -
圖 1(A). 實驗模型與假說。 - 52 -
圖 1(B).實驗設計。 - 53 -
圖2. K562細胞的白血病幹細胞亞群的特徵表現。 - 54 -
圖 3. K562母細胞、CD34+/CD34+與CD34+/CD38-兩個白血病幹細胞亞群的訊號表現。 - 55 -
圖 4. K562母細胞、CD34+/CD34+與CD34+/CD38-兩個LSCs亞群在Histone Deacetylases的訊號表現。 - 56 -
圖 5 比較CD34+/CD38− and CD34+/CD38+ LSCs對Iimatinib抗藥性的表現。 - 57 -
圖 6. CD34+38-白血病幹細胞經HDACi治療後訊號表現。 - 58 -
圖 7. CD34+38-經IM與SAHA治療後細胞存活率表現。 - 59 -
圖 8. CD34+38-經IM與SAHA合併治療後訊號表現。 - 60 -
圖 9. 使用FACS方法觀察CD34+38-經IM與SAHA單獨或合併治療後的細胞凋亡情況。 - 61 -
第八章 附圖 - 62 -
附圖1.CML抗藥性基因表現 - 63 -
附圖2.CML抗藥性基因表現的Reference Marker - 63 -
第九章 參考文獻 - 64 -

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