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研究生: 楊佳芳
Yang, Jia-fang
論文名稱: 利用糞便菌叢移植探討pterostilbene及其衍生物3'-hydroxypterostilbene對高脂飲食誘發肥胖小鼠腸道發炎與菌叢失調之影響
Effects of pterostilbene and its derivative 3'-hydroxypterostilbene on intestinal inflammation and dysbiosis in obesity mice induced by high-fat diet using fecal microbiota transplantation
指導教授: 陳與國
Chen, Yu-Kuo
學位類別: 碩士
Master
系所名稱: 農學院 - 食品科學系所
Department of Food Science
論文出版年: 2023
畢業學年度: 111
語文別: 中文
論文頁數: 76
中文關鍵詞: 肥胖糞便菌叢移植菌叢失調腸道菌相
外文關鍵詞: Obesity, fecal microbiota transplantation, dysbiosis, gut microbiota
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6346/NPUST202300088
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  • 近年來,隨著飲食習慣逐漸西式化、精緻飲食增加及作息不正常,過多的能量逐漸堆積體內,導致脂肪組織的增生,使世界上罹患肥胖的人口正逐年上升,並且肥胖會提高其他併發症的發生機率,而根據研究指出,腸道中的腸道菌群之失調與人體的肥胖有一定的相關性。紫檀芪 (Pterostilbene, Pt) 和其衍生物3'-hydroxypterostilbene (OHPt) 是一種天然存在的芪類化合物,來源於多種天然植物,像是葡萄、花生及藍莓等,具有極高的生物活性,並在肥胖老鼠中有改善其腸道菌相的功用。故本研究為利用糞便菌叢移植模式探討Pt及其衍生物OHPt對於肥胖小鼠及腸道發炎反應的影響。本實驗組別總共分為7組:C組:基礎飲食;C-Pt組:基礎飲食+50 mg/kg/day Pt;C-OHPt組:C+50 mg/kg/day OHPt;HFD組:高脂飲食組;HFD-F-C組:高脂飲食組+糞便來自C組小鼠;HFD-F-Pt組:高脂飲食組+糞便來自C-Pt組小鼠;HFD-F-OHPt組:高脂飲食組+糞便來自C-OHPt組小鼠。實驗結果顯示,HFD-F-OHPt組小鼠血清中總膽固醇及三酸甘油酯含量明顯低於HFD-F-C組及HFD-F-Pt組,且在肝臟中脂質的累積也有明顯的降低,與HFD組相比具有顯著性差異 (p<0.05)。除此之外,在發炎指標性物質LPS方面亦具有降低發炎效果,並在西方墨點法中的數據顯示HFD-F-OHPt組降低發炎蛋白的表現優於HFD-F-C組及HFD-F-Pt組。根據以上結果可以得知,HFD-F-OHPt組的處理對於因肥胖而造成血管及肝臟的脂肪堆積,具有改善的效果,並能降低因肥胖而造成的發炎反應,且改善效果較優於HFD-F-C組及HFD-F-Pt組,後續實驗將會分析其糞便菌相,探討其改善肥胖之關鍵菌種,做為抗肥胖的益生質或益生菌保健食品之開發。

    In recent years, with the gradual westernization of eating habits, exquisite diet and abnormal work and rest, too much energy gradually accumulates in the body, leading to the proliferation of adipose tissue, making the world's obese population increasing year by year, and obesity will increase the incidence of complications. According to the research, it is pointed out that the imbalance of intestinal flora in the intestine is related to obesity.
    Red sandalwood stilbene and its derivative 3'- hydroxypterostilbene red sandalwood stilbene are natural stilbene compounds derived from a variety of natural plants, such as grapes, peanuts and blueberries. They have high bioavailability and can improve the intestinal microflora of obese mice. Therefore, the purpose of this study was to investigate the effects of pterostilbene and its derivatives on obese mice and intestinal inflammation by using the fecal flora transplantation model. The experimental group is divided into 7 groups:C group:basic diet, C-Pt group:basic diet +50 mg/kg/day pterostilbene, C-OHPt group:basic diet +50 mg/kg/day 3'-hydroxypterostilbene, HFD group:high-fat diet group, HFD-F-C group:high-fat diet group + feces from group C mice, HFD-F-Pt group:high-fat diet group + feces from group C-Pt mice, HFD-F-OHPt group:high-fat diet group +feces from group C-OHPt mice. According to the experimental results, the HFD-F-OHPt group decreased the total cholesterol and triglyceride in serum better than the HFD-F-C group and the HFD-F-Pt group, and significantly reduced the lipid accumulation in the liver. Compared with the HFD group, the HFD-F-OHPt group had a significant difference, and the inflammatory index substance LPS had a lower inflammatory effect. The data in the Western ink dot method showed that the HFD-F-OHPt group decreased the inflammatory response better than the HFD-F-C group and the HFD-F-C group. According to the above results, HFD-F-OHPt group can improve the fat accumulation in blood vessels and liver caused by obesity, and reduce the inflammatory reaction caused by obesity, and the improvement effect is better than that of HFD-F-Pt group and HFD-F-C group. Subsequent experiments will analyze the bacteria in feces, and explore the key bacteria to improve obesity, as a prebiotic or probiotic health food development for anti-obesity.

    壹、前言 1
    貳、文獻回顧 2
    一、肥胖 (Obesity) 2
    二、非酒精性脂肪肝病 (Nonalcoholic fatty liver disease) 3
    (一)、非酒精性脂肪肝炎介紹 3
    (二)、脂質累積 4
    (三)、胰島素 (Insulin) 4
    (四)、胰島素抵抗 (Insulin resistance, IR) 4
    三、腸道菌相 (Gut microbiota) 6
    四、糞便菌叢移植 (Fecal microbiota transplant, FMT) 7
    五、脂多醣 (Lipopolysaccharide, LPS) 8
    六、腸道發炎相關路徑 9
    (一)、Nuclear factor-κB (NF-κB) 9
    (二)、COX-2 9
    (三)、誘導型一氧化氮合酶 (Inducible nitric oxide synthase, iNOS) 10
    (四)、IL-6 10
    (五)、腫瘤壞死因子α (TNF-α) 10
    七、紫檀芪 (Pterostilbene, Pt) 與3'-羥基紫檀芪(3'-hydroxypterostilbene, OHPt) 11
    參、材料與方法 21
    一、實驗架構圖 21
    二、實驗材料與儀器設備 22
    (一)、實驗樣品 22
    (二)、化學藥品與試劑 22
    (三)、血液生化分析試紙 25
    (四)、動物實驗飼料和油品 26
    (五)、酵素套組 26
    (六)、儀器設備 26
    三、實驗方法 28
    (一)、實驗動物飼養 28
    四、實驗動物犧牲 30
    (一)、血液 30
    (二)、肝臟 31
    (三)、脂肪 31
    (四)、大腸與盲腸 31
    (五)、腎脾 31
    (六)、犧牲血液測試 32
    五、肝臟分析 32
    (一)、製備肝臟萃取液 32
    六、糞便菌相分析 33
    (一)、萃取糞便DNA及菌相分析 33
    七、病理組織切片 33
    (一)、製備石蠟切片 33
    (二)、H&E 染色 (H&E stain) 34
    八、西方墨點法 34
    (一)、大腸蛋白質液製備 34
    (二)、定量蛋白質濃度 34
    (三)、樣品配製 35
    (四)、聚丙烯醯胺膠體電泳 (SDS-PAGE electrophoresis) 35
    九、統計分析 37
    肆、結果與討論 38
    一、Pt和OHPt糞便菌叢移植對肥胖老鼠之攝食及體重影響 38
    二、Pt和OHPt糞便菌叢移植對肥胖老鼠之脂肪組織影響 39
    三、Pt和OHPt糞便菌叢移植對肥胖老鼠之血清生化值影響 40
    四、Pt和OHPt糞便叢移植對肥胖老鼠腸道病理分析 43
    五、Pt和OHPt糞便叢移植對肥胖老鼠腸道相關蛋白影響 44
    六、菌相分析 44
    伍、結論 46
    陸、圖表資料 47
    柒、參考文獻 67

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