臺灣博碩士論文加值系統

現今癌症是人類主要的死因之一，因此許多的科學家一直都在尋找和發展能夠有效預防或抑制癌症生長的藥物與方法。我們利用液態發酵培養虎奶菇菌絲體，收集其胞外多醣，並且在經過 DEAE 陰離子與膠體層析純化後，我們取得一部分多醣 3BN-L，其導致 SW620 結腸癌細胞株 caspase 3 活性上升與 PRAP 的剪切，Bcl-xL蛋白的表現量亦顯著減少，並且使細胞週期 G2/M 期轉換蛋白 cyclin B1、Cdc25c、CDK1 蛋白表現量下降。3BN-L 誘使 SW620 細胞發生細胞凋亡，並可觀察到 JNK 與 p38 MAPK 訊息傳遞路徑活化與細胞內 ROS 含量的增加。加入 ROS 的阻斷物 NAC，3BN-L 誘發的細胞死亡情形則被減緩。我們發現細胞內 ROS 含量的增加與虎奶菇胞外多醣的抑癌活性有著十分密切的關聯。此外，給予異體移植 SW620 細胞的裸鼠 3BN-L (180 mg/kg/d i.p.) 四週，相對於只給予 PBS 的組別，腫瘤大小有將近 50 % 的抑制。結果顯示3BN-L 引起的細胞凋亡可能是透過 JNK 與 p38 MAPK 路徑的活化與細胞內 ROS 產生量增加，這些是導致 SW620 結腸癌細胞株死亡的一個可能機制。

Colon cancer represents a major cause of cancer related morbidity and mortality. To reduce this tremendous health burden, new approaches have been directed toward extremes of the disease spectrum centering on strategies for colon cancer prevention and therapy. Treatment of SW620 cells with 3BN-L resulted in activation of the caspase 3 and cleavage of poly ADP-ribose polymerase (PARP); whereas, the expression of Bcl-xL were decreased markedly. The expressions of G2/M phase transition proteins - cyclin B1, Cdc25c and CDK1 also down regulation. 3BNL-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK and p38 MAPK, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed 3BNL-induced apoptosis. Furthermore, 3BN-L (180 mg/kg/d i.p.) reduced the growth of SW620 xenografts in nude mice by ~50 % at 4 weeks compared with PBS groups (p < 0.01). These results leads us to speculate that 3BN-L may play an apoptotic cascade by activation of the JNK/p38 pathway and induction of ROS production, providing a proposed mechanism for 3BN-L- induced cell death in human colon cancer cells.

目錄 i
中文摘要 iv
英文摘要 v
第一章 緒論 1
第一節 菇類多醣的抗腫瘤活性 1
第二節 虎奶菇 2
第三節 細胞週期 3
第四節 細胞凋亡 (apoptosis) 4
一、 細胞凋亡現象 4
二、 細胞凋亡訊息傳遞 4
第五節 DNA fragmentation 與 sub-G1 細胞 5
第六節 Reactive oxygen species (ROS) 5
第七節 逆境反應/壓力反應 (stress response) 相關激酶路徑 6
一、 Mitogen-activated protein kinase (MAPK) 6
二、 c-Jun N-terminal kinase (JNK) 6
三、 p38 mitogen-activated protein kinases (p38 MAPK) 7
第八節 環境壓力的感受器 ASK1 激酶 7
第九節 研究動機與實驗設計 8
第十節 參考資料之附圖 10
第二章 實驗材料與方法 14
第一節 實驗材料 14
一、 虎奶菇菌種 14
二、 細胞株 14
三、 藥品 14
四、 抗體 15
五、 藥品配製 15
六、 儀器設備 17
七、 動物 17
第二節 實驗方法 18
一、 深層液態培養菌絲體胞外水萃多醣之製備 18
二、 樣品中糖含量之測定 21
三、 蛋白質濃度之測定 21
四、 細胞培養 21
五、 細胞存活度 22
六、 細胞形態觀察與 DAPI 染色 22
七、 細胞週期及 sub-G1 細胞比例之測量 22
八、 Annexin V 細胞凋亡測量法 23
九、 Caspase 3 活性之測定 24
十、 Western Blot 25
十一、 細胞內 ROS 含量之測量 26
十二、 動物與腫瘤的異體移植 (xenograft) 27
第三章 實驗結果 28
第一節 虎奶菇菌絲體外多醣的純化 28
第二節 3BN-L 的處理對 SW620 細胞形態上的影響 28
第三節 透過 3BN-L 所引起的細胞週期停滯與細胞凋亡現象 28
第四節 3BN-L 活化逆境反應激酶並降低 G2/M 轉換蛋白表現 29
第五節 3BN-L 的處理造成細胞內 ROS 產生 29
第六節 3BN-L 的處理抑制異體移植 SW620 腫瘤的生長 30
第四章 討論 31
第一節 虎奶菇菌絲體深層液態培養胞外多醣的新穎性與優點 31
第二節 多醣的純化 31
第三節 虎奶菇菌絲體深層液態培養胞外多醣抗腫瘤的可能機制 32
第四節 未來與展望 33
第四章 參考文獻 34
第五章 附圖與表格 39
表一 虎奶菇菌體外水萃多醣不同純化階段各部份之比較 39
表二 3BN-L fraction對 SW620 細胞其細胞週期細胞分布比例之影響 39
表三 3BN 經凝膠過濾層析分析後，各 fraction 之佔有比例以及醣類、蛋白組成比例之比較 40
圖一、虎奶菇菌絲體深層液態發酵培養胞外水萃多醣之純化流程圖 41
圖二、利用 dihydroethidium (DHE) 偵測活細胞內 ROS 含量之原理 42
圖三、SW620 細胞異體移植動物實驗流程圖 43
圖四、DEAE 陰離子交換層析之結果、中性及酸性多醣萃取物對 SW620 細胞其細胞存活度之影響 44
圖五、膠體過濾層析分析中性多醣萃取物之結果、膠體過濾層析中性多醣萃取物之活性部分 45
圖六、3BN-L fraction 對 SW620 細胞其細胞與細胞 DNA 片斷化現象之影響 46
圖七、3BN-L fraction 對 SW620 細胞其 sub-G1 細胞比例之影響 47
圖八、3BN-L fraction 對 SW620 細胞凋亡細胞比例之影響 48
圖九、3BN-L 的處理對 SW620 細胞 caspase 3 活性之影響 49
圖十、3BN-L 的處理對細胞週期 G2/M 週期轉換所需蛋白表現量及 apoptotic marker顯現之影響 50
圖十一、3BN-L 的處理對壓力反應激酶訊息傳遞路徑活化之影響 51
圖十二、3BN-L 的處理對 SW620 細胞內 ROS 量之影響 52
圖十三、NAC 的處理對由 3BN-L 造成的細胞內 ROS 量增加與生長抑制現象之影響 53
圖十四、3BN-L 對異體移植於裸鼠之 SW620 腫瘤生長大小之影響 54
圖十五、給予 3BN-L 之組別與對照組間腫瘤生長大小之比較 55
圖十六、推測 3BN-L 誘使 SW620 細胞發生細胞凋亡及 G2/M 細胞週期停滯之可能機制 56

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