臺灣博碩士論文加值系統

癌症位於十大死因之首，因著飲食文化與環境的改變，大腸癌發生率
也逐年攀升，目前大腸癌主要的治療方法主要為手術、化放療，以及
標靶治療。香杉芝(Antrodia salmonea，AS)是一種木材腐朽型真菌，為
台灣特有真菌，具有抗發炎、抗氧化、抗腫瘤等作用；目前研究尚未
有香杉芝作用在大腸癌的機制探討。本實驗目的分為兩個部分，一為
香杉芝水萃物在小鼠發炎化學致癌模式之大腸致癌預防之探討，第二
部分為香杉芝水萃物在大腸癌細胞(SW620, HT29, HCT116)中是否透過
誘導細胞凋亡及細胞自噬以及其相關機制來達到抗癌功效。第一部分
的動物實驗結果顯示，香杉芝水萃物會改善小鼠發炎化學致癌模式
Azoxymethane (AOM) / Dextran sodium sulfate (DSS)之腫瘤形成。而在
第二部分的細胞實驗結果顯示，香杉芝對大腸癌細胞具有細胞毒性，
給予 AS 會增加促凋亡蛋白及抗凋亡蛋白 Bax/ Bcl2 ratio 的表現以及下
游蛋白 caspase3 以及 PARP 的裂解，在 Annexin V/PI 雙染實驗中亦可
發現 AS 會誘導大腸癌細胞的凋亡。而在細胞自噬方面的實驗結果顯
示，AS 會促進自噬相關蛋白 P62、LC3 以及降低 Bcl2、Atg4B 蛋白的
表現，亦可促進酸性囊泡的生成。此外，AS 誘導之細胞凋亡及細胞自噬之關係，經時間點及抑制劑 3-Methyladenine (3-MA)、ZVAD-fmk 的
實驗結果顯示，AS 在大腸癌 SW620 細胞中會先促進保護性自噬的發
生來抑制細胞凋亡，待細胞自噬逐漸減弱時細胞凋亡才開始發生，而
其中的相關機制可能是透過抑制 AKT/mTOR 以及 NF-κB 入核，或者是
透過活化 ERK 路徑來影響自噬及凋亡之發生。AS 的給予會誘導
Reactive oxygen species (ROS)的生成，在給予 ROS 抑制劑 N
acetylcysteine (NAC) 後可顯著改善 AS 對大腸癌細胞存活率的影響，
並減少細胞凋亡以及細胞自噬的發生，表示 AS 會透過增加 ROS 的生
成來誘導細胞凋亡及細胞自噬的發生，進而影響大腸癌的細胞存活。

The incidence of colorectal cancer has increased yearly due to the change in dietary culture and environment. At present, surgery, chemotherapy and target treatment are the main regimen for colorectal cancer. Antrodia salmonea (AS), a medicinal mushroom in Taiwan, has an extensive range of biological activities, including anti-inflammatory, antioxidant and anti-tumor etc. In the current study we separate the experiments into two part, the first is to investigated whether Antrodia salmonea (AS) can prevent Azoxymethane (AOM) / Dextran sodium sulfate (DSS)–induced tumor formation, the second part is to clarify whether induce apoptosis and autophagy to prevent human colon cancer cell line (SW620, HT29, HCT116). The result demonstrated that AS has great potential to suppress colon tumorigenesis and also to improve AOM/DSS-induced colonic shortening of ICR mice. The findings showed that AS is cytotoxic to colon cancer cell line SW620, HT29, HCT116 with IC50 values 169.6, 235.1, 163.2 μg/mL respectively. Results from western blot, and Annexin V/PI double stain showed that AS induced apoptosis in SW620 cells, and pretreatment of cells with caspase inhibitor Z-VAD-fmk disminished ASinduced apoptotic cell death. AS also induced autophagy in SW620 cells, proved by upregulation of autophagy related protein (LC3, P62) in SW620 cells, followed by increased formation of acidic vesicular organells (AVO) with AS treatment. To know the induction of autophagy by AS in SW620 cells is for death or for survival, pretreatment of cells with autophagy inhibitors (3MA or CQ) significantly increased the AS-induced apoptotic cell death. Further to clarify the upstream mechanism, our results showed that AS induce protective autophagy by increasing phosphorylation of ERK and downregulating AKT/mTOR signaling pathway. AS can also induce apoptosis by downregulating AKT/mTOR signaling pathway and inhibiting NF-κB nuclear translocation. In addition, AS triggered Reactive oxygen species (ROS) production and pretreatment of cells with ROS inhibitor Nacetylcysteine (NAC) suppressed AS-induced apoptotic cell death and protective autophagy. These results conclude that AS induced apoptosis and protective autophagy in SW620 cells via ROS production.

目錄
目錄 1
圖目錄 4
縮寫表 5
摘要 6
Abstract 8
前言 9
文獻探討 11
第一節 香杉芝 12
背景介紹 12
相關藥理研究 13
相關抑癌研究 13
第二節 大腸癌 15
大腸癌介紹 15
大腸癌症狀 15
大腸癌分類 15
大腸癌危險因子 15
大腸癌診斷 17
大腸癌分期 17
大腸癌治療方式 18
大腸癌之形成 19
大腸癌相關基因表現 20
發炎與大腸癌形成之關係 24
大腸癌之動物誘導模式 25
第三節 大腸癌細胞分類 28
第四節 細胞凋亡 29
第五節 細胞自噬 32
第六節 細胞凋亡及自噬與癌症的相關性 34
第七節 活性氧 (Reactive oxygen species, ROS) 35
第八節 活性氧與細胞凋亡的關係 36
第九節 活性氧與細胞自噬的關係 37
研究動機 38
實驗架構 39
第一部分 動物實驗 39
第二部分 細胞實驗 40
材料與方法 41
第一節 實驗儀器 42
第二節 實驗材料 43
耗材 43
藥品 44
一級抗體 46
二級抗體 47
第三節 實驗方法 48
細胞實驗 48
香杉芝水萃物製備 48
細胞培養 48
解凍細胞 49
繼代培養 49
細胞冷凍 50
細將胞型態(Cellular morphology) 50
細胞存活率試驗(MTT assay) 50
細胞總蛋白質萃取(total protein extraction) 52
抽取核質蛋白 54
蛋白質定量 56
西方墨點法(Western blot) 57
活性氧 (ROS，Reactive Oxygen Species)生成測定 62
酸性囊泡胞器試驗 (Acridine Orange assay) 63
Annexin V/PI staining 64
統計分析 65
腸癌動物誘導實驗 66
動物與試劑 67
實驗方法與步驟 68
小鼠組織樣品採集(血、肝、腎、脾、大腸) 69
腸中腫瘤之數目及大小 69
組織蛋白質萃取 70
組織包埋及切片 71
免疫化學組織染色(immunochemistry Staining) 72
GOT(AST)&GPT(ALT) 75
實驗結果與圖表 76
第一部分 77
第一節 77
第二節 77
第三節 78
第二部分 85
第一節 AS對大腸癌細胞之存活率影響 85
第三節 AS誘導人類大腸癌細胞發生細胞自噬 91
第四節 AS誘導細胞凋亡與細胞自噬之相關性 93
第五節 AS透過MAPK(ERK1/2)訊息傳遞路徑對細胞誘導之凋亡及自噬影響 101
第六節 AS對人類大腸癌細胞PI3K/AKT路徑之影響 104
第七節 AS促使大腸癌細胞產生活性氧。 106
討論 110
第一部分 動物致癌模式之預防實驗 111
第二部分 112
AS對大腸癌細胞之存活率影響 112
AS在大腸癌細胞株SW620中所誘導之細胞凋亡以及細胞自噬的關係 112
AS在大腸癌細胞中透過MAPK(ERK1/2)路徑影響細胞凋亡及細胞自噬 114
AS 對人類大腸癌細胞 PI3K/AKT 路徑之影響 114
AS 促使大腸癌細胞產生活性氧 115
結論 116
參考文獻 117
圖目錄
圖.1 Heme Iron from Meat and Risk of Colorectal Cancer: A Meta- analysis and a Review of the Mechanisms Involved 16
圖.2 AOM / DSS誘導小鼠大腸癌腫瘤進展的示意圖 19
圖.3結直腸癌（CRC）相關的遺傳性綜合徵 20
圖.4MOLECULAR DEFECTS IN COLORECTAL TUMORIGENESIS 21
圖.5 Wnt/β-Catenin signaling pathway 22
圖.6 The RAS-RAF-MEK-ERK Signaling Cascade in NSCLC. 23
圖.7 Mechanisms of colorectal cancer (CRC) and colitis-associated cancer (CAC) development 24
圖.8 Metabolism of AOM involves multiple transcriptionally regulated xenobiotic-metabolizing enzymes. 26
圖.9 Gene mutations and altered expression of proteins in rat colon carcinogenesis induced by AOM. 27
圖.10 Colon cancer cell line origins 28
圖.11 PI3K/AKT mediated signaling promotes cell survival 31
圖.12 Effector proteins of Kirsten rat-sarcoma (KRAS) and apoptosis. 31
圖.13 The classical autophagy pathway proceeds through a series of well-defined steps. 32
圖.14.Schematic representation on apoptotic effects of morin on HCT-116 human colon cancer cells. 36

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