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研究生:李昱陞
研究生(外文):LEE-YU-SHENG
論文名稱:白樺茸誘導人類結腸直腸癌HCT-116細胞凋亡
論文名稱(外文):Inonotus obliquus induces apoptosis in human colorectal carcinonma HCT-116 cells.
指導教授:蔡政志蔡政志引用關係
學位類別:碩士
校院名稱:弘光科技大學
系所名稱:食品科技所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:147
中文關鍵詞:白樺茸結腸直腸癌NF-κBCOX-2細胞周期G0/G1期停滯粒線體凋亡路徑
外文關鍵詞:Inonotus obliquuscolorectal cancerNF-κBCOX-2cell cycle G1/S phase arrestmitochondrial apoptotic pathway
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中文摘要
由於國人飲食、作息不正常,近年來結腸直腸癌發生率及死亡率每年呈快速攀升的趨勢。藥用菇白樺茸含有許多活性成份,具有治療腸胃道疾病、癌症、及結核病等疾病之功效。本篇研究探討白樺茸液態發酵菌絲體抑制結腸直腸癌細胞 HCT-116 的增殖及活性,利用 MTT 進行細胞存活率分析,並以乳酸脫氫酶(Lactic dehydrogenase ; LDH)釋放量判定細胞膜的完整性。促凋亡及抑制凋亡的介質mRNA表現量以即時定量聚合酶鏈鎖反應(Real-time PCR) 進行分析,其中 P53 及 NF-κB 蛋白質含量以西方墨點法分析,並利用酵素連結免疫分析法(enzyme-linked immunoadsorbent assay ; ELISA) 分析細胞激素 IL-4、IL-6、IL-8、IL-12(p40)、IL-10、IFN-γ及 TNF-α 含量。也同時評估白樺茸菌絲體對 HCT-116 細胞Caspase-3 活性及一氧化氮(NO)含量之影響。結果顯示:(1)粒線體凋亡路徑:白樺茸菌絲體可提升促凋亡基因(Bax, Bad, TNFSF10, Caspase-3)及Bax/Bcl-2 比率。此外,20mg/mL 白樺茸使 HCT-116 細胞之 Caspase-3 活性增加。 (2)誘導細胞周期 G0 期到 G1 期的停滯:白樺茸可增進促凋亡基因(p53, p21WAF1/CIP1)及抑制抗凋亡基因(CyclinD1)之 mRNA 表現,並可增加 HCT-116 細胞之 p53 蛋白質表現量。 (3)細胞激素: 即時定量聚合酶鏈鎖反應結果顯示,可降低抗凋亡細胞激素IL-6及IL-8 mRNA表現量。此外,酵素連結免疫分析法定量的結果顯示,白樺茸菌絲體可降低 IL-4、IL-6、IL-8 及 IL-12濃度。(4)白樺茸菌絲體減少了 HCT-116 細胞之 NF-κB 蛋白質表現量及 COX-2 mRNA表現量。綜合上述結果,白樺茸菌絲體可能可作為一種新的改善結腸直腸癌的保健素材。
關鍵字: 白樺茸;結腸直腸癌; NF-κB ; COX-2;細胞周期G0/G1期停滯;粒線體凋亡路徑。

Abstract
Because of irregular dietary habits and life style in Taiwan, the incidence and mortality rate of colorectal cancer have been increasing rapidly these year. The medicinal mushroom Inonotus obliquus (I. obliquus) contains several active ingredient, and it can be effective to cure cancer, digestive system diseases, tuberculosis and other illnesses. This study investigated the inhibitory activity against the proliferation of human colorectal cancer HCT-116 cells by I. obliquus obtained from submerged fermentation. Cell viability were measured by the reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT) and cell membrane integrity were determined by lactic dehydrogenase ( LDH) release. The mRNA expression of proapoptosis and antiapoptosis mediators was assayed by real-time PCR, and the levels of p53 and NF-κB were assessed using Western blot analysis, and levels of cytokine IL-4, IL-6, IL-8, IL-12 (p40), IL-10, IFN-γ and TNF-α were quantified by Enzyme-linked immunosorbent assay (ELISA) to analyse. Furthermore, the influence of I.obliquus to HCT-116 cells were evaluated by caspase-3 activity and nitric oxide (NO) production. The results can be summerised as:(1) the mitochondrial apoptotic pathway: Quantiative RT-PCR data showed up-regulation for proapoptotic genes (bax, bad, tnfsf 10, caspase-3) and increased bax/bcl-2 ratio by I.obliquus. Moreover, treating with 20mg/mL I.obliquus augmented caspase-3 activity in HCT-116 cells. (2)Induction of cell cycle G0/G1 phase arrest: I. obliquus up-regulated the mRNA expression of proapoptotic genes (p53, p21WAF1/CIP1) and down-regulated antiapoptotic gene (CyclinD1), while I. obliquus mycelia increased the expressions of p53 protein in HCT-116 cells. (3)Cytokines : Quantitative RT-PCR results showed down-regulation of antiapoptotic genes IL-6 and IL-8, and ELISA results showed down-regulation of IL-4, IL-6, IL-8 and IL-12. (4) I.obliquus decreased the expression of NF-κB protein and COX-2 gene in HCT-116 cells. Taking together, I.obliquus may be used as a potentially novel food material for health care to improve the treatment of colorectal cancer.


Keyword: Inonotus obliquus;colorectal cancer;NF-κB;COX-2;cell cycle G1/S phase arrest;mitochondrial apoptotic pathway.

目錄
頁碼
中文摘要 I
Abstract II
目錄 IV
表次 VII
圖次 VIII
第一章、文獻整理 1
第一節、藥用菇 1
第二節、白樺茸 2
一、白樺茸學名 2
二、白樺茸的生長環境 2
三、白樺茸的功效及應用 2
第三節、白樺茸生理活性成分 4
第四節、結直腸癌 9
一、結直腸癌 9
二、結腸直腸癌發生原因 10
三、結直腸癌分期 12
四、世界各國結直腸癌概況 14
五、藥物抑制癌細胞之方法 14
第五節、凋亡相關路徑 15
一、細胞週期調控 15
二、細胞激素及其他物質與結腸直腸癌之關係 20
第六節、粒線體凋亡路徑 29
第七節、結腸直腸癌治療用藥5-FU 33
第二章、前言 34
第三章、材料與方法 36
一、實驗菌株及細胞株來源 36
(一)白樺茸菌株 36
(二)腸道細胞株 36
(一)細胞培養基 37
(二) 藥品 38
(三)實驗套組 39
三、實驗方法 40
(一) 細胞培養 40
(二)細胞存活率分析(MTT) 40
(三) LDH分析 42
(四) Total RNA之萃取 43
(五) 即時定量聚合酶連鎖反應 (Real-time Quantitative Polymerase Chain Reaction ) 44
(六) Caspase-3活性分析 45
(七) 酵素連結免疫分析法 (Enzyme-linked immunosorbent assay ; ELISA) 46
(八)西方墨點法 (Western blot assay) 47
(九) 白樺茸抑制發炎能力評估 (Nitric Oxide) 55
(十) 統計分析 56
四、實驗儀器設備 60
第四章、結果與討論 62
一、 經白樺茸菌絲體處理的結直腸癌細胞存活率以及膜完整性分析………..………………………………………………….62
二、白樺茸菌絲體調控結腸直腸癌細胞周期 63
三、白樺茸菌絲體影響粒線體凋亡相關基因 65
四、白樺茸菌絲體調節凋亡相關細胞激素 67
五、白樺茸菌絲調節NF-κB蛋白質量及COX-2基因表現量 …………………………………………………..70
六、白樺茸菌絲體抑制發炎相關激素產生 71
第五章、結論 73
第六章、參考文獻 117

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