臺灣博碩士論文加值系統

在罹患結腸直腸癌病人中除了手術切除外，在化療、標靶治療及放射性化療通常令患者放棄療程。然而，這些類型的治療通常帶來相當大的副作用，導致患者生理和心理上的痛苦，甚至惡化病人的健康狀況。本項研究的目的是探討從小孢子靈芝所分離出來的真菌免疫調節蛋白（Ganoderma microsporum immunomodulatory protein , GMI）對HCT-15結腸直腸癌細胞的毒殺作用以及其導致細胞凋亡的可能機制。初步實驗結果發現隨著GMI濃度的增加，HCT-15細胞的細胞形態明顯改變，也發現GMI會增加HCT-15細胞活性氧物質(ROS)的產生，同時表現出活化的Caspase 3、Caspase 7、PARP，最後導致細胞凋亡。此外，GMI與5-FU的聯合治療的細胞實驗中揭示GMI具有拮抗作用，有效殺死HTC-15癌細胞。同時使用GMI可以降低5-FU的劑量需求以毒殺HTC-15癌細胞。我們利用MTAMs的小鼠模式與斑馬魚模式進行體內實驗，發現GMI也會誘導HCT-15細胞死亡，然而此濃度下的GMI並不會損傷小鼠器官或組織之病變。除了老鼠動物模式外，GMI 對斑馬魚胚胎發育的影響，亦被評估。結果顯示，GMI 濃度高於0.5M對斑馬魚胚胎發育是有影響的。總結，GMI有可能是具治療大腸癌的重組免疫蛋白藥物之一。

Surgery is always the first option to treat various forms of solid cancers, such as colorectal cancers (CRCs). Following surgical removal of tumors from patients with CRCs, chemotherapy, targeted therapy or radiotherapy will normally give in to patients. However, those therapeutic managements usually bring enormously adversary side effects, leading to unpleasant physiological and psychological suffering in patients or even to deteriorate patients’ health condition. Therefore, to find the alternative therapeutic candidates with fewer side effects is encouraged. In this study, we would like to evaluate the effects of GMI, a novel constituent isolated from the leaves of Ganoderma microsporum, on cell proliferation of human colorectal cancer HCT-15 cells and then unbraid the underlying GMI-mediated potential killing mechanisms. Our preliminary data showed that GMI alter the changes in cell morphology of HCT-15 cells. We also noticed that GMI induced cell stress by increased reactive oxygen species (ROS) production from HCT-15 cells. Furthermore, GMI also decreased cell survival and proliferations resulted in the activations of Caspase 3, Caspase 7, PARP, leading to DNA fragmentation coupled with cell apoptosis. In addition to sole treatment, we also evaluated the therapeutic effects of the combined treatments of GMI plus 5-FU on CRCs. The results suggested that combined treatment displayed more effectively to inhibit HTC-15 cell growth with lower the dosage requirement of 5-FU. Furthermore, we also evaluate the therapeutic effects of GMI on MTAMs animal models and zebrafish. The results demonstrated that the numbers of HCT-15 cells in MTAM chambers were reduced after exposure to GIMS. Moreover, no cytotoxic effects were observed by examinations of animal organs and tissues. In the fish model, we noticed that the concentration of GMI more than 0.5M GMI possessed toxicity in zebrafish embryonic development experiments. Taking all together, it suggests that GMI is a potential candidate for treatment of colorectal cancers in patients but its cytotoxic mechanism needs to be further characterized.

目　　　錄
目錄 I
圖次目錄 III
表次目錄 IV
縮寫表 V
摘要 VI
Abstract VIII
第一章　緒論 1
第二章　文獻探討 3
第一節　何謂結腸直腸癌 4
第二節　靈芝的藥理研究與成分發現 5
第三節　粒線體與細胞凋亡 9
第四節　活性氧物質和細胞凋亡的關係 12
第五節　實驗目的 13
第三章　實驗材料與方法 14
第一節　實驗材料 15
第二節　實驗方法 21
第四章　實驗結果 30
第一節　探討GMI對HCT-15細胞的毒殺性及死亡途徑 31
第二節　探討GMI在動物體裡的抗癌效果及毒性測試 34
第五章　結論與討論 35
第六章 參考文獻 40
第七章 圖表 44
附表一 56
附表二 58
附表三 59

圖　次　目　錄
圖2-1 GMI的三級結構，由四個單體組合而成 8
圖2-2 LZ-8、G. Tsugae和G. microsporum序列 8
圖2-3 LZ-8、金針菇免疫調節蛋白(FIP-fve)和GMI的立體構造 9
圖2-4研究設計架構圖 13
圖3-1 MTT試劑還原示意圖 16
圖3-2老鼠實驗設計圖示 28
圖6-1 HCT-15細胞在經過GMI不同濃度處理後的形態 45
圖6-2 GMI對HCT-15細胞的毒殺情形 46
圖6-3 GMI誘導HCT-15細胞的凋亡 47
圖6-4 GMI誘導HCT-15細胞內產生ROS的情形 48
圖6-5 GMI對HCT-15細胞內caspase -3,-7、PARP蛋白表現 49
圖6-6 GMI與 5-FU對HCT-15細胞毒殺效果 50
圖6-7 GMI對於MTAMs模式小鼠治療體重及肝臟重量變化 51
圖6-8小鼠背後皮下血管分布分析 52
圖6-9斑馬魚形態與存活率 55

表　次　目　錄
表6-1小鼠血液生化分析 53
表6-2小鼠全血液分析 54

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