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  1. NTU Theses and Dissertations Repository
  2. 生物資源暨農學院
  3. 食品科技研究所
Please use this identifier to cite or link to this item: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91950
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???org.dspace.app.webui.jsptag.ItemTag.dcfield???ValueLanguage
dc.contributor.advisor潘敏雄zh_TW
dc.contributor.advisorMin-Hsiung Panen
dc.contributor.author林偉盛zh_TW
dc.contributor.authorWei-Sheng Linen
dc.date.accessioned2024-02-27T16:11:05Z-
dc.date.available2024-02-28-
dc.date.copyright2022-04-26-
dc.date.issued2022-
dc.date.submitted2002-01-01-
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91950-
dc.description.abstract結腸直腸癌 (colorectal cancer, CRC) 罹癌率於全世界及我國常年位居前三,其致病機制與飲食有著密不可分之關係,其中雜環胺 (heterocyclic amines, HCAs) 為肉類、水產品等富含蛋白質食物經高溫處理所產生,而 PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) 為人類飲食中暴露量相較高之雜環胺,且毒性資料最為完整,多項流行病學指出其對腸癌之發展有極高的攸關性。值得注意的是,現今藉由飲食預防病症之觀念逐漸重視,如許多膳食調查與流行病學指出大蒜之攝取可減少罹患大腸癌之風險,然而受限於強烈的辛辣味及刺激感不宜直接食用,因此大蒜之加熱熟成、發酵等加工製品近年受到關注,包括黑蒜。黑蒜已被證實較生蒜具有更高之生物活性,如抗氧化、抗發炎及神經保護等,歸因於其具更高含量之植化素,如硫烯丙基半胱胺酸 (S-allylcysteine, SAC),先前的研究顯示 SAC 與其他有機硫化物 (organosulfur compounds) 相比,具有較低之毒性與副作用,並於體內外試驗中發現具調升抗氧化酵素相關因子與抑制發炎受體與下游路經進而減少氧化壓力與抑制發炎反應,此外也發現具減緩癌細胞的增生與促凋亡之活性,擁有癌症化學預防之潛力,因此本研究建立以 PhIP/DSS 誘導小鼠腸癌之模式,探討 SAC 是否具減緩食安汙染物雜環胺所造成之腸癌發展。結果顯示,於飲食中每天給予 0.05% SAC 可抑制 PhIP/DSS 誘導小鼠之結腸長度縮短和結腸瘜肉數目的形成。此外,補充SAC可以顯著抑制 PhIP/DSS 誘導的血漿和結腸組織促炎細胞因子,並調降結腸組織中 iNOS、COX-2 及 MMP-2 蛋白表現,並阻止腸道屏障 E-cadherin 蛋白表現量下降。此外,SAC 可能藉由上調結腸組織中 pERK 1/2/Nrf2信號傳導來增加 HO-1 之表達,進而降低氧化壓力水平,而抑制 PhIP 對起始期細胞之 DNA 損害與促進期發炎反應,減輕 PhIP/DSS 引起的結腸炎與腫瘤形成。此外,透過 16S rRNA 定序分析糞便檢體腸道菌相,結果顯示 PhIP/DSS 組別具有較高腸炎與腸癌相關菌群如 Erysipelotrichaceae、Bifidobacteriaceae 及 Clostridiaceae 與較低抑制發炎相關菌群 Lachnospiraceae;而在有給予 SAC 組別有較低之可能使雜環胺活化菌屬 Bacteriodes,顯示 SAC 可改善腸癌情況之腸道微生物穩態,使其與正常飲食組相似。而為探究 SAC 是否抑制 PhIP 引起的早期基因損傷並減少癌起始細胞形成,以體外模式評估 SAC 對經 PhIP 處理之人類正常結腸粘膜上皮細胞 NCM 460 之影響,顯示 PhIP 誘導會造成氧化壓力失衡與 DNA 損傷,而透過蛋白質體外結合實驗與細胞熱轉移分析法,顯示 SAC與 Keap1 蛋白可能具有親和力關係;此外,SAC 也可能透過抑制 p38 與增加ERK1/2與AKT之磷酸化進而降低 Keap1 與 Nrf2 之結合,使 Nrf2/HO-1 信號上調而增加 GSH與GSH/GSSG 比值,進而抑制PhIP 誘導之 ROS 上升與 DNA 損傷。此外 SAC 可抑制 AhR、ARNT、CYP1A1 及 CYP1B1 蛋白質表現,顯示可能具避免致癌物代謝轉化作用之潛力存在。綜合上述,顯示 SAC 可能抑制 PhIP 誘導正常腸細胞之氧化壓力失衡與 DNA 損傷,並減輕小鼠腸道中之發炎反應,進而減緩 PhIP/DSS 誘導的結直腸癌發生,闡明黑蒜成分中 SAC 的癌症化學預防試劑的理論基礎及影響方式,期可作為輔助腸癌化學預防之方案,並作為未來相關產業功能性食品開發之依據。zh_TW
dc.description.abstractColorectal cancer (CRC) has the third-highest incidence and the second-highest mortality of all cancers worldwide. Worthwhile, CRC is closely related to dietary and environmental factors. In particular, a high intake of processed and red meat increases the risk of CRC, as the high-temperature cooking process induces the formation of mutagenic and carcinogenic compounds, such as polycyclic aromatic hydrocarbons and heterocyclic amines (HCAs) compounds. One of these compounds, namely, 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine (PhIP), is the most abundant HCAs formed in cooked fish, poultry, and meat. Chemopreventive phytochemicals can block or reverse the premalignant stage in multistep carcinogenesis or retard precancerous cells into malignant cells. Thus, chemoprevention is a promising strategy for preventing cancer. Many dietary surveys and epidemiological studies have pointed out that garlic intake can reduce the risk of CRC. However, due to its strong spicy taste and revolting flavor, it is not suitable to be eaten directly. Therefore, processed products such as aging or fermented product have attracted attention in recent years, including black garlic. Previous studies have demonstrated S-allylcysteine (SAC) is a natural organosulfur compound that has been studied extensively, and SAC is relatively high amounts in black garlic. Numerous recent studies have revealed that SAC has low toxicity and possesses various biofunctional properties, including anti-oxidation, anti-inflammatory, and retard cancer cell proliferation in vivo and in vitro. Therefore, we want to explore whether SAC could prevent carcinogenesis caused by food-contaminants HCAs. The results show that 0.05% SAC dietary supplementation significantly reduced colon shortening and tumor formation by up-regulating pERK1/2/Nrf2/HO-1 antioxidant signaling, which suppressed pro-inflammatory mediators such as iNOS, COX-2, and MMP-2 in the colon mucosa. Moreover, SAC supplementation alleviated intestinal barrier disruption. In addition, 16S rRNA sequencing was used to analyze the gut microbiota of fecal samples, and the results showed that PhIP/DSS group had higher colitis and colon cancer-related flora such as Erysipelotrichaceae, Bifidobacteriaceae, and Clostridiaceae and lower anti-inflammation bacteria Lachnospiraceae. Moreover, the SAC treatment group had lower Bacteriodes levels. These bacteria may lead to a strong increase in the bacterial mutagenicity of HCAs. The principal component analysis plot showed that SAC-supplemented group were closely clustered around that of the control group, suggesting that SAC has a marked direct or indirect effect on the composition of the gut microbial community and also reversed PhIP/DSS-induced gut dysbiosis. Furthermore, we investigate the inhibitory effect of SAC against NCM 460, a normal human colon mucosal epithelial cell line, carcinogenesis caused by PhIP during the initiation stage of carcinogenesis. The result shows that SAC may have an affinity with Keap1 protein using pull-down assay and cellular thermal shift assay. SAC may also reduce the binding of Keap1 and Nrf2 by inhibiting p-p38 and increasing the phosphorylation of ERK1/2 and AKT, thereby increasing the Nrf2/HO-1 signal up-regulated the ratio of GSH to GSH/GSSG, which inhibits the PhIP-induced oxidative stress and DNA damage. In addition, SAC can inhibit the expression of AhR, ARNT1, CYP1A1, and CYP1B1 proteins level, showing that it may potentially avoid the metabolic transformation of carcinogens. Overall, this is the first investigation with evidence of SAC’s ability to mitigate PhIP-induced ROS production and DNA damage and block inflammatory signaling at PhIP/DSS-induced colitis hence retard cancer initiation and promotion. These results indicate the potential application of SAC in colon cancer chemoprevention.en
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dc.description.tableofcontents口試委員會審定書 I
誌謝 II
摘要 V
Abstract VII
目錄 IX
附圖目錄 XIII
附表目錄 XIV
圖目錄 XV
縮寫表 XVII
前言 1
第一章、文獻回顧 2
第一節、結腸直腸癌 (Colorectal cancer) 2
(一)、結腸直腸癌流行病學概況 2
(二)、結腸直腸癌概述 4
(三)、發炎反應與結腸直腸癌發展之關係 5
(四)、腸道菌相對結腸直腸癌發展之影響 7
第二節、雜環胺 (Heterocyclic amine, HCAs) 10
(一)、潛藏於食品中之危害因子 10
(二)、雜環胺概述 12
(三)、雜環胺之致癌性 14
(四)、2-胺基-1-甲基-6-苯基咪唑[4,5-b]吡啶(2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, PhIP) 危害風險與致癌機制 15
第三節、食物中植物天然化合物於癌症化學預防策略 17
(一)、天然物於癌症化學預防簡介 17
(二)、植化素之癌症化學預防策略 19
第四節、硫烯丙基半胱胺酸 (S-allylcysteine, SAC) 27
(一) 、硫烯丙基半胱胺酸簡介 27
(二) 、硫烯丙基半胱胺酸生物活性 28
第二章、研究目的與實驗架構 32
第一節、研究目的 32
第二節、實驗架構 33
第三章、材料方法 34
第一節、實驗材料與儀器 34
(一)、樣品與誘導劑 34
(二)、細胞株 34
(三)、試藥與耗材 34
(四)、抗體 36
(五)、儀器 37
第二節、動物實驗 (in vivo) 38
(一)、實驗動物品系與飼養 38
(二)、實驗動物分組 38
(三)、動物試驗方法 38
(四)、體重、攝食及飲水測量 39
(五)、動物犧牲與臟器觀察 39
(六)、血清生化值分析 40
(七)、蘇木精-伊紅與免疫組織化學染色 40
(八)、細胞激素與趨化因子含量測定 47
(九)、腸道菌相分析 48
(十)、腸道組織萃取 50
第三節、細胞試驗 (in vitro) 51
(一)、細胞株培養 51
(二)、細胞存活率試驗 53
(三)、一氧化氮之測定 54
(四)、氧化壓力測定 55
(五)、麩胱甘肽、麩胱甘肽/氧化型麩胱甘肽比值測定 55
(六)、DNA 萃取與片段化分析 57
(七)、慧星試驗 (comet assay) 59
(八)、蛋白質體外結合實驗 (pull down assay) 61
(九)、細胞熱轉移分析法 (cellular thermal shift assay) 63
(十)、細胞轉染與小髮夾RNA基因減弱 64
(十一)、細胞蛋白質萃取 65
(十二)、蛋白質定量 66
(十三)、蛋白質電泳與西方墨點法 67
第四節、統計分析 70
第四章、結果與討論 71
第一節、評估 SAC 抑制 PhIP/DSS 誘導 ICR 小鼠腸癌之功效 71
(一)、外觀、體重、攝食量及飲水量之變化 71
(二)、黑蒜成分 SAC 改善 PhIP/DSS 誘導小鼠的臨床體徵 73
(三)、SAC 減輕 PhIP/DSS 誘導腸癌小鼠結腸長度縮短與腫瘤病變 75
第二節、黑蒜成分 SAC 抑制體外與體內試驗之發炎反應 78
(一)、SAC 抑制 LPS 誘導小鼠單核巨噬細胞 Raw 264.7 之發炎反應 78
(二)、SAC 抑制 PhIP/DSS 誘導小鼠腸癌模式之發炎反應 79
第三節、黑蒜成分 SAC 上調抗氧化相關蛋白並抑制 PhIP/DSS 誘導腸道屏障受損 80
第四節、黑蒜成分 SAC 改善 PhIP/DSS 誘導小鼠腸道菌相組成 85
第五節、以體外模式評估 SAC 對 PhIP 誘導 NCM 460 之影響 90
(一)、不同濃度之 SAC 與 PhIP 對 NCM 460 細胞存活率之影響 90
(二)、SAC 抑制 PhIP 誘導 NCM 460氧化壓力失衡 90
(三)、SAC對 PhIP 誘導 NCM 460細胞DNA 損傷之影響 95
(四)、SAC對 PhIP 誘導 NCM 460細胞質Keap1與核內Nrf2蛋白質水平之影響並探討 SAC 與 Keap1 親和力關係 99
(五)、SAC 對 PhIP 誘導 NCM 460 細胞之 MAPKs與 PI3K/AKT 路徑之影響 104
(六)、SAC對 PhIP 誘導 NCM 460 細胞 AhR/CYP450 路徑相關蛋白之影響 107
第五章、結論與展望 110
第六章、參考文獻 112
已發表之國際期刊論文 131
附錄 133
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dc.language.isozh_TW-
dc.subject結腸直腸癌zh_TW
dc.subject雜環胺zh_TW
dc.subject葡聚醣硫酸鹽zh_TW
dc.subject硫-烯丙基半胱胺酸zh_TW
dc.subject癌症化學預防zh_TW
dc.subject腸道菌相zh_TW
dc.subjectdextran sulfate sodiumen
dc.subjectcolorectal canceren
dc.subjectmicrobiotaen
dc.subjectcancer chemopreventionen
dc.subjectS-allylcysteineen
dc.subjectheterocyclic aminesen
dc.title以 PhIP 及 DSS 誘導模式探討 S-allylcysteine 對腸癌之化學預防功效zh_TW
dc.titleStudying chemopreventive effects of S-allylcysteine on colon carcinogenesis induced by PhIP and DSS modelen
dc.typeThesis-
dc.date.schoolyear110-2-
dc.description.degree博士-
dc.contributor.oralexamcommittee吳明賢;廖秀娟;何元順;楊登傑;王應然;黃步敏;郭靜娟;張嘉哲zh_TW
dc.contributor.oralexamcommitteeMing-Hsien Wu;Vivian Hsiu-Chuan Liao;Yuan-Soon Ho;Deng-Jye Yang;Ying-Jan Wang;Bu-Miin Huang;Ching-Chuan Kuo;Chia-Che Changen
dc.subject.keyword結腸直腸癌,雜環胺,葡聚醣硫酸鹽,硫-烯丙基半胱胺酸,癌症化學預防,腸道菌相,zh_TW
dc.subject.keywordcolorectal cancer,heterocyclic amines,dextran sulfate sodium,S-allylcysteine,cancer chemoprevention,microbiota,en
dc.relation.page133-
dc.identifier.doi10.6342/NTU202200634-
dc.rights.note未授權-
dc.date.accepted2022-03-17-
dc.contributor.author-college生物資源暨農學院-
dc.contributor.author-dept食品科技研究所-
Appears in Collections:食品科技研究所

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