人類第9 號染色體與第22 號染色體發生轉位之後產生費城染色體(Philadelphia chromosome)，因而產生具有致癌性的融合蛋白BCR-ABL。BCR-ABL 為持續活化的酪胺酸激酶(tyrosine kinase)，會啟動下游許多關於細胞增生、存活、以及自我更新的訊號傳遞，而導致細胞惡性轉型。分子量為190 kD 的BCR-ABL 融合蛋白多發現於急性淋巴性白血病(ALL) (Ph+ ALL)，其疾病惡性度高，預後不佳；現階段以最高強度化學治療搭配tyrosine kinase inhibitors (TKI,如: Imatinib, Dasatinib)抑制BCR-ABL的活性，雖可使病人獲得暫時性緩解，但是仍有高比例病人血癌復發，並且發生抗藥性。因此，發展有別於化療的藥物或與tyrosine kinase inhibitor 合併搭配使用，是治療Ph+ ALL 的重要課題。10￠(Z),13￠(E),15￠(E)-heptadecatrienyl
hydroquinone (HQ17(3))是萃取自漆樹的天然小分子，對於多種腫瘤細胞具有毒殺能力，但對於人類正常周邊血液單核細胞以及實驗鼠無明顯毒性，HQ17(3)在腫瘤細胞中具有抑制DNA 拓樸異構酶II (DNA topoisomerase II)以及誘導產生reactiveoxygen species (ROS)能力，並可在肝癌細胞株中引起DNA 損傷及apoptotic cell death。我們在前驅測試中發現以低濃度HQ17(3)處理帶有BCR-ABL 的ALL 細胞株-SUP-B15 24 小時即有顯著毒殺作用。
本研究結果顯示HQ17(3)會誘導SUP-B15 細胞活性氧(ROS)上升、酸性胞器出
現、粒線體膜電位喪失、染色體斷裂及細胞自噬標記LC3-II 產生。抗氧化劑
(Antioxidantss, ROS scavenger)穀胱苷肽(GSH)與維生素C 可以減緩HQ17(3)所誘導的ROS 產生、減少粒線體膜電位損傷並降低細胞死亡。親脂性排鐵劑desferrioxamine mesylate (DFO)可阻止酸性胞器產生，並阻斷HQ17(3)所引起的粒線體損傷及細胞死亡；加入抑制細胞自噬抑制劑3methyl adenine (3-MA)與氯奎寧(Chloroquine)亦可以減少HQ17(3)誘發之細胞死亡。綜合本研究的實驗結果顯示HQ17(3)誘導ROS 啟動細胞自噬可能是造成SUP-B15 細胞死亡重要原因，並且此過程高度依賴鐵存在。因此，本研究結果指出，若可誘導癌細胞產生細胞自噬將有潛力發展輔助治療帶費城染色體急性淋巴性白血病的策略。

Reciprocal t(9;22); BCR-ABL translocation gives rise to Philadelphia(Ph)chromosome and results in production of chimeric BCR-ABL fusion protein with constitutively active tyrosine kinase activity. The BCR-ABL protein activates a number of signaling pathways which promote cell proliferation, survival and self-renewal. Ph+ acute lymphoblastic leukemia (ALL) with 190kD BCR-ABL fusion protein presents very poor clinical outcomes. Although tyrosine kinase inhibitor (TKI) combined with multi-agent chemotherapy help to acquire a complete remission temporarily, still a high proportion of patients have leukemic relapse and develop drug resistance. Therefore,
developing drug(s) to act in different ways as in chemotherapies, or to be used in combination with the TKI in treatment of Ph+ ALL, is an alternative way to help the
patients affected by this very high risk disease. 10￠(Z),13￠(E),15￠(E)-heptadecatrienyl hydroquinone (HQ17(3)) is a small natural molecule extracted from the R. succedanea.
DNA topoisomerase IIα inhibition and oxidative stress were found to account for selective cytotoxicity caused by HQ17(3) in various types of tumor cells. In the preliminary data, we found that HQ17(3) has significant cytotoxic effect in a p190 BCR-ABL Ph+ ALL cell line, SUP-B15, within 24 hours in micromolar concentration.
We found in SUP-B15 cells, HQ17(3) induced reactive oxygen species (ROS) and acidic vesicle formation, mitochondrial membrane potential disturbance, chromosome breakage, and emergence of an autophagy marker, cleaved LC3-II. ROS scavengers(Glutathione, vitamin C) attenuated HQ17(3)-induced cell injury. Lysosomotropic iron chelator, desferrioxamine mesylate (DFO) abolished HQ17(3)-induced acidic vesicles formation, mitochondrial membrane potential loss and cell death. Inhibitors for autophagy (3-methyl adenine, chloroquine) partially rescued cells from
HQ17(3)-induced death. These results indicated that HQ17(3) may induce ROS production, and subsequently lead to autophagic cell death. In conclusion, HQ17(3) displayed a significant anti-leukemic activity in Ph+ ALL (SUP-B15) cells by ROS production and lysosomal iron-dependent events that contribute to autophagic cell death. These results suggest that agents selectively induce ROS in leukemic cells might induce autophagic cell death, and would potentially augment the treatment for Ph+ ALL.

致謝I
中文摘要II
AbstractIV
縮寫表VI
第一章緒論1
第一節急性淋巴性白血病1
1.1 急性淋巴性白血病的症狀1
1.2 急性淋巴性白血病1
1.3 急性淋巴性白血病的診斷2
1.4 急性淋巴性白血病的治療2
1.5 急性淋巴性白血病的預後因子4
1.6 費城染色體陽性急性淋巴性白血病簡介4
1.7 費城染色體5
1.8 費城染色體陽性急性淋巴性白血病治療5
1.9 以IM 為基底的治療在Ph+ ALL 所面臨的問題6
第二節對苯二酚衍生物HQ17(3)簡介7
2.1 對苯二酚的代謝7
2.2 對苯二酚相關文獻探討7
2.3 DNA 拓樸異構酶(DNA topoisomerases II, Topo II)8
2.4 對苯二酚衍生物-HQ17(3)相關文獻探討8
第三節活性氧分子reactive oxygen species9
3.1 活性氧分子reactive oxygen species9
3.2 ROS 與細胞死亡11
第四節計畫性細胞死亡(Programmed cell death)12
4.1 細胞死亡定義12
4.2 細胞凋亡(Apoptosis, type I cell death)12
4.3 計畫性細胞壞死(Necroptosis)14
4.4 由Lysosome(溶小體)主導的細胞死亡方式15
4.4.1 Lysosome 的介紹15
4.4.2 Lysosomal membrane permeabilization (LMP)15
4.4.3 LMP 的偵測16
4.5 細胞自噬(Autophagy, type II cell death)17
4.5.1 autophagic cell death17
4.5.2 autophagy 的偵測19
第二章研究目的與實驗設計20
第一節研究目的20
1.1 研究目的20
1.2 研究動機20
第二節實驗設計21
第三章材料與方法22
第一節實驗材料22
1.1 細胞株22
1.2 試藥/劑、抗體、儀器、耗材清單22
1.3 各式溶液及其配方26
1.3.1 細胞培養、繼代26
1.3.2 流式細胞儀相關實驗26
1.3.3 萃取細胞蛋白質27
1.3.4 鈉十二烷基硫酸鹽聚丙烯胺凝膠電泳與膠體轉漬27
1.3.5 西方墨點法30
1.3.6 細胞存活/活性分析31
1.3.7 Sub-G1 分析 (chromosome fragmentation 分析)32
第二節實驗方法 32
2.1 解凍細胞、細胞培養及細胞計數32
2.2 細胞活性測試及藥物IC50 33
2.2.1 trypan blue exclusion assay33
2.2.2 ACP assay 33
2.3 PS 磷脂質外翻特徵與細胞膜完整性分析 (Annexin V/PI stain)33
2.4 細胞粒線體膜電位的測定(DiOC6(3) stain)34
2.5 Chromosome DNA fragmentation assay (sub-G1 fraction in DNA content analysis)35
2.6 細胞內caspase 活性分析(西方墨點法 35
2.6.1 蛋白質萃取 35
2.6.2 蛋白質定量、稀釋與電泳樣品準備 36
2.6.3 鈉十二烷基硫酸鹽聚丙烯胺凝膠電泳與膠體轉漬 (SDS-PAGE and
blotting)36
2.6.4 阻斷非特異性結合與免疫染色36
2.7 螢光染色 37
第四章結果38
第一節 HQ17(3)對於SUP-B15 細胞具有毒性 38
1.1 SUP-B15 對於imatinib 有抗性38
1.2 HQ17(3)對於SUP-B15 細胞具有毒性 38
1.3 HQ17(3)對於正常血球細胞不具毒殺能力39
第二節 HQ17(3)處理細胞後，SUP-B15 出現死亡特徵 39
2.1 HQ17(3)促使SUP-B15 細胞膜脂質外翻、細胞膜受損39
2.2 HQ17(3)處理細胞後，SUP-B15 細胞出現粒線體膜電位的喪失 40
第三節 HQ17(3)處理細胞後，引起ROS 相關路徑40
3.1 HQ17(3)誘導SUP-B15 細胞產生ROS40
3.2 抗氧化劑可以減少HQ17(3)所造成的細胞損傷 41
第四節 HQ17(3)引起caspase-independent cell death42
4.1 HQ17(3)僅誘導微量的caspase 3活化 42
4.2 pan-caspase inhibitor 無法挽救細胞死亡 42
第五節 HQ17(3)與Necroptosis 42
5.1 Necrostatin-1 無法挽救細胞死亡42
5.2 同時處理z-VAD 與Nec-1 無法挽救細胞死亡43
第六節 HQ17(3)可誘導酸性胞器表現 43
6.1 HQ17(3)處理細胞後酸性胞器增多 43
6.2 HQ17(3)可造成iron-dependent 細胞死亡 44
6.3 鐵螯合劑可減少酸性胞器產生 44
6.4 Lysosome 內酸性水解酶非導致SUP-B15 死亡原因45
6.5 HQ17(3)誘導細胞產生autophagy 45
6.6 抑制autophagy 可挽救部分細胞死亡46
6.7 DFO 抑制autophagy 產生46
第五章討論 48
第六章參考文獻 53
圖與表 59
附錄 82

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