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研究生:廖婉如
研究生(外文):Wan-Ru Liao
論文名稱:玉米赤黴烯酮對秀麗隱桿線蟲老化之毒性效應及相關機制探討
論文名稱(外文):Evaluation of Toxic Effects of Zearalenone on Aging and the Underlying Mechanisms in Caenorhabditis elegans
指導教授:魏嘉徵
指導教授(外文):Chia-Cheng Wei
口試委員:廖秀娟潘敏雄王如邦林怡君
口試委員(外文):Hsiu-Chuan LiaoMin-Hsiung PanReu-Ben WangYi-Jun Lin
口試日期:2021-01-26
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品安全與健康研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:58
中文關鍵詞:食品安全黴菌毒素玉米赤黴烯酮老化秀麗隱桿線蟲
外文關鍵詞:food safetymycotoxinzearalenoneagingCaenorhabditis elegans
DOI:10.6342/NTU202100555
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近年來臺灣人民對於食品安全相關議題日漸關注,如何保障國人食之安全亦是相當需要重視的議題之一。而食品中黴菌毒素之汙染便是急需關注的食品安全議題。過去玉米赤黴烯酮之相關研究主要針對其生殖毒性之探討,而在生物體老化及相關機制之研究則相對較少,且過往研究主要以高濃度暴露玉米赤黴烯酮為主要研究設計,但在日常飲食中未必能暴露至如此高劑量,故本篇研究設計將貼近實際暴露情形,利用秀麗隱桿線蟲 (Caenorhabditis elegans) 為活體模式生物,探討較低濃度長期暴露玉米赤黴烯酮對生物體老化之影響,並進一步探討其分子調控相關機制。本研究分別就C. elegans之體長、運動行為 (body bends與head thrashes) 及體內活性氧化物質 (reactive oxygen species, ROS) 累積情形進行毒性評估,並依實驗結果挑選較具代表性之暴露濃度進行與老化相關之毒性效應及相關機制探討。實驗結果發現,暴露0.3、1.25、5、10及50 μM玉米赤黴烯酮至成蟲第0天,並不會影響C. elegans之體長,但上述濃度均能顯著對C. elegans之運動行為造成負面影響,且顯著增加C. elegans體內ROS之累積。當暴露1.25 及50 μM 玉米赤黴烯酮至成蟲第6或第8天,皆能顯著降低C. elegans之咽喉收縮率並對運動行為造成負面影響;暴露50 μM 玉米赤黴烯酮至成蟲第4、6或8天能顯著提高C. elegans體內ROS及脂褐素累積量、延長C. elegans之排泄週期,且玉米赤黴烯酮能明顯減少C. elegans壽命。同時,在相關機制探討部分發現,將daf-16 mutant暴露於1.25 及50 μM 玉米赤黴烯酮其咽喉收縮率之下降幅度高於野生種,且暴露1.25 及50 μM玉米赤黴烯酮會抑制DAF-16的入核。進一步的探討暴露50 μM玉米赤黴烯酮對於老化相關基因表達的影響發現,暴露至成蟲第0天時,hsp-16.1、hsp-16.49、hsp-70、daf-16及sod-3的基因表達量顯著上升,而ctl-1及ctl-2的表達量則顯著下降;暴露至成蟲第4天的結果則發現,hsp-16.49、hsp-70及sod-3的表達量顯著下降,而ctl-1及ctl-2的表達量亦顯著下降。綜合本研究之實驗結果,玉米赤黴烯酮的暴露,確實會促進C. elegans之老化,並影響insulin/IGF-1 signaling pathway轉錄因子DAF-16及其下游基因之表達。
Food safety issues have increasingly been a concerned topic in Taiwan in recent years. Among them, the contamination of mycotoxins in food needs urgent attention. Previous zearalenone related research has mainly focused on its reproductive toxicity. Studies related to its effects on aging and the underlying mechanism are relatively limited. In addition, previous studies were mostly conducted under high exposure concentrations of zearalenone. However, such high concentrations may not be a point of exposure in our daily diet. To reflect the actual exposure scenario, this present study aimed to investigate the toxic effects of long-term exposure to lower concentrations of zearalenone on aging and the underlying mechanism by using Caenorhabditis elegans as an in vivo model organism. We observed body length, locomotion (body bends and head thrashes), the accumulation of reactive oxygen species (ROS), aging markers, and investigated the aging-related mechanism after zearalenone exposure. The results showed that exposure to 0.3, 1.25, 5, 10 and 50 μM zearalenone did not affect the body length of C. elegans, but the above concentrations significantly reduced the body bends and head thrashes, and increased the accumulation of ROS in C. elegans. In addition, exposure to 1.25 and 50 μM zearalenone to day 4, 6, or 8 of adulthood significantly reduced the pharyngeal pumping rate and locomotion of C. elegans. Further, exposure to 50 μM zearalenone significantly increased the accumulation of ROS and lipofuscin, reduced the pharyngeal pumping rate, extended the defecation cycle, and shortened the lifespan of C. elegans. Moreover, exposure to 1.25 and 50 μM zearalenone further decreased the pumping rate of daf-16 mutant compared to N2, and inhibited the translocation of DAF-16 into the nucleus in C. elegans. We further explored the effects of exposure to 50 μM zearalenone on the expression of aging-related genes. The results showed that the expression levels of hsp-16.1, hsp-16.49, hsp-70, daf-16, and sod-3 genes were significantly increased, while the expression level of ctl-1 and ctl-2 were decreased on day 0 of adulthood C. elegans after 50 μM zearalenone exposure. Furthermore, the expression levels of hsp-16.49, hsp-70, and sod-3 genes were then significantly decreased on day 4 of adulthood C. elegans after 50 μM zearalenone exposure. The expression levels of ctl-1 and ctl-2 genes were also significantly decreased on day 4 of adulthood C. elegans after 50 μM zearalenone exposure.
In conclusion, the exposure of zearalenone accelerated the aging process via the insulin/IGF-1 signaling pathway by inhibiting DAF-16 translocation and affecting downstream genes expression.
碩士學位論文口試委員會審定書 i
誌謝 ii
摘要 iii
Abstract iv
Graphic abstract v
Highlights vi
目錄 vii
圖次 ix
表次 x
1. 研究動機 1
2. 文獻回顧及研究目的 3
2.1玉米赤黴烯酮 (zearalenone) 3
2.2 Zearalenone之汙染近況與相關規範 3
2.3 Zearalenone之生物毒性 7
2.3.1 Zearalenone之吸收、分布、代謝及排除 7
2.3.2 Zearalenone之基因毒性 7
2.3.3 Zearalenone之肝毒性 8
2.3.4 Zearalenone之免疫毒性 8
2.3.5 Zearalenone之致癌毒性 9
2.3.6 Zearalenone之生殖毒性 9
2.4 以秀麗隱桿線蟲 (Caenorhabditis elegans) 探討zearalenone之慢性暴露
毒性 10
2.4.1 秀麗隱桿線蟲 10
2.4.2 以秀麗隱桿線蟲探討老化 11
2.5 研究目的 12
3. 材料與方法 14
3.1實驗架構流程圖 14
3.2實驗藥品 15
3.3 C. elegans品系與培養條件 15
3.4 Zearalenone對C. elegans生長之毒性測試 15
3.5 Zearalenone對C. elegans運動行為之毒性測試 15
3.6 Zearalenone影響C. elegans體內過氧化物質 (ROS) 累積試驗 16
3.7 Zearalenone影響C. elegans體內脂褐素 (lipofuscin) 累積試驗 16
3.8 Zearalenone對C. elegans老化相關行為之毒性試驗 17
3.9 Zearalenone對C. elegans生命週期影響試驗 (life span assay) 18
3.10 DAF-16入核分析試驗 (DAF-16 translocation assay) 18
3.11定量即時聚合酶鏈鎖反應(qRT-PCR) 19
3.12統計分析 19
4. 結果與討論 20
4.1 暴露zearalenone對C. elegans體長之影響 20
4.2 暴露zearalenone對C. elegans運動行為之影響 22
4.3 暴露zearalenone對C. elegans體內活性氧化物質之影響 24
4.4 暴露zearalenone對C. elegans體內脂褐素 (lipofuscin) 累積情形之影響 28
4.5 暴露zearalenone對C. elegans老化相關行為指標之影響 29
4.6 暴露zearalenone對C. elegans壽命 (lifespan) 之影響 34
4.7 暴露zearalenone對C. elegans DAF-16之影響 36
4.8 暴露zearalenone對C. elegans老化相關基因表達量之影響 41
5. 結論 44
6. 參考文獻 45
7. 附錄 55
附錄一、臺灣對於食品中zearalenone之限量標準 55
附錄二、引子序列 56
附錄三、Zearalenone對E. coli OP50生長之影響 57
附錄四、Two-way ANOVA 58
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