臺灣博碩士論文加值系統

根據行政院衛生署民國九十七年的統計資料發現，大腸癌高居國人十大癌症死亡比例的第二位；而大腸癌一般好發年齡大多聚集於五十歲以上的患者。而在大腸癌的臨床治療方法上，許多的化療藥物大多對於正常的組織部分具有高毒殺作用，對於病患往往會產生不良的副作用，因此，研發有效且對於正常組織具有低毒殺效果的抗癌化療藥物乃是目前刻不容緩的事情。
為了開發天然藥物於癌症治療上的應用，我們由台灣特有蕨類「粗毛金星蕨」的甲醇萃取物中，分離出新的黃酮類化合物WYC02，且經由活性測試結果得知，WYC02具有抗癌效果。WYC02-9係由實驗室所合成之WYC02的衍生物；在人類大腸癌細胞株HC116的毒殺效果中，WYC02-9比WYC02具有更顯著的毒殺性。在本研究中，藉由細胞群落分析及軟瓊脂培養方式發現 WYC02-9能有效抑制癌細胞的生長。細胞週期方面，了解WYC02-9會讓癌細胞週期停滯於G2/M期。而在細胞凋亡部分，發現WYC02-9會誘導活化caspase-8、caspase-9、caspase-3以及PARP，致使癌細胞走向細胞凋亡；在活體動物實驗發現，WYC02-9不僅能有效抑制腫瘤的生長，甚至對於裸鼠的體重、造血功能及肝、腎功能都無產生不良的副作用。WYC02-9亦會活化p38 MAPK表現，而利用抑制p38 MAPK的表現，可發現確實會造成癌細胞的存活比例上升，藉由結果可了解 p38 MAPK對於WYC02-9誘導癌細胞凋亡中佔有一席之地。另一方面，藉由西方墨點法及電子顯微鏡觀察等方式得知WYC02-9會誘導LC3-Ⅱ蛋白表現，造成細胞自噬的產生。

According to the data of Department of Health R.O.C. report, colorectal cancer (CRC) is the second leading cause of cancer death in Taiwan, and most of the cases occur after 50 old. Many chemotherapeutic agents used in clinics have high toxicity to normal tissues and cause many side-effects on patients. Therefore, it is important to search for effective and low toxic anticancer drugs for cancer therapy.
WYC02 is a novel flavonoids isolated from the whole plant of T. torresiana and has a potent anticancer effect. WYC02-9, a derivative of WYC02, shows a more potent cytotoxicity than WYC02 on HCT116 colorectal cancer cells. We identified that WYC02-9 inhibited the cancer cells growth by colony formation and soft agar assays. In nude mice model, WYC02-9 had a significant anti-cancer activity against colorectal tumor growth without major side effects. Moreover, WYC02-9 arrested cell cycle progression at G2/M phase. And, WYC02-9 forced cancer cells to go through apoptosis by inducing the activity of caspase-8, caspase-9, caspase-3, and inhibiting the activity of PARP. WYC02-9 inhibited cell proliferation by promoting the activity of p38 MAPK, which was reversed by p38 MAPK inhibitor, suggesting that p38 MAPK may play a major role in WYC02-9-induced cell death. On the other hand, WYC02-9 induced LC3-Ⅱ protein, and then induced autophagy in cancer cells.

中文摘要I
英文摘要II
誌謝III
目錄IV
表目錄VI
圖目錄VII
第一章 研究背景1
1-1 大腸癌(Colorectal cancer)1
1-2 細胞凋亡(Apoptosis)2
1-3 細胞凋亡訊息傳遞路徑 (Apoptosis signaling pathway)2
1-4 細胞自噬(Autophagy)4
1-5 黃酮類化合物(Flavonoids)6
第二章 研究動機9
2-1 研究動機9
2-2 實驗架構11
第三章 研究方法與材料12
3-1 細胞培養12
3-2 細胞計數法13
3-3 細胞增生試驗13
3-4 細胞形態學觀察14
3-5 細胞群落分析15
3-6 軟瓊脂培養法15
3-7 細胞週期分析16
3-8 Annexin V-FITC細胞凋亡分析17
3-9 電子顯微鏡觀察18
3-10 SB203580及3-Methyladenine(3-MA)之抑制劑處理19
3-11 西方墨點法19
3-12 裸鼠動物實驗22
3-13 免疫組織染色23
第四章 研究成果與討論24
4-1 研究成果24
4-1-1 WYC02-9抑制大腸癌細胞增生與細胞群落形成24
4-1-2 WYC02-9抑制大腸癌細胞週期的進行25
4-1-3 WYC02-9引起大腸癌細胞進行細胞凋亡作用25
4-1-4 WYC02-9活化大腸癌細胞株HCT116中p38 MAPK活性26
4-1-5 p38 MAPK在WYC02-9引起的生物活性上所扮演的角色26
4-1-6 WYC02-9在活體實驗中，藉由產生細胞凋亡作用及活化p38 MAPK而抑制腫瘤的生長27
4-1-7 WYC02-9引起大腸癌細胞株HCT116進行細胞自噬作用28
4-1-8 Autophagy在WYC02-9引起的生物活性上所扮演的角色28
4-2 成果圖表30
4-3 成果討論47
第五章 結論與未來展望50
第六章 參考文獻52
表目錄
Table 1. IC50 of novel flavonoid compounds and oxaliplatin on colorectal cancer cells30
Table 2. Complete blood count and biochemical profile for the nude mice after treatment with WYC02-9 for 6 weeks42
圖目錄
附圖一 細胞凋亡傳導路徑7
附圖二 MAPK pathway 7
附圖三 PI3K/AKT pathway 8
附圖四 粗毛金星蕨、WYC02及WYC02-9結構圖10
附圖五 實驗流程圖11
附圖六 細胞凋亡的型態變化14
附圖七 軟瓊脂培養基示意圖16
附圖八 磷脂絲胺酸(PS)外翻現象17
Figure 1. Effects of WYC02-9 on inhibiting the growth of HCT116 colorectal cancer cells 31
Figure 2. The effects of WYC02-9 on colony formation of HCT116 cells 32
Figure 3. The effect of WYC02-9 on soft agar colony formation of HCT116 cells 33
Figure 4. The effects of WYC02-9 on cell cycle distribution of HCT116 colorectal cancer cells 34
Figure 5. Protein involved in G2/M progression were modulated by WYC02-9 35
Figure 6. WYC02-9 induced apoptotic cell death in HCT116 colorectal cancer cells 36
Figure 7. Proteins involved in apoptosis pathway upon WYC02-9 treatment 37
Figure 8. WYC02-9 enhanced the activation of MKK 3/6 and p38 MAPK in HCT116 colorectal cancer cells 38
Figure 9. SB203580 reversed the WYC02-9 induced activation of p38 MAPK and growth inhibition in HCT116 colorectal cancer cells 39
Figure 10. SB203580 reversed the WYC02-9 induced activation of p38 MAPK and growth inhibition in HCT116 colorectal cancer cells by western blot 40
Figure 11. WYC02-9 suppressed HCT116 colorectal tumor growth in nude mice xenograft assay 41
Figure 12. The cleavage PARP and p-p38 MAPK expression in colorectal tumor tissues 43
Figure 13. Proteins involved in autophagic maker were regulated by WYC02-9 44
Figure 14. WYC02-9 induces autophagy in HCT116 colorectal cancer cells 45
Figure 15. 3-MA reversed WYC02-9 induced autophagy and growth inhibition in HCT116 colorectal cancer cells 46

中文部份
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英文部份
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