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研究生:陳宣智
研究生(外文):Chen, Hsuan-chih
論文名稱:降低斑馬魚MicroRNA-27b以增加脂肪生成之功能性研究
論文名稱(外文):Functional role of MicroRNA-27b depletion increased zebrafish lipogenesis
指導教授:何國牟
指導教授(外文):Her, Guro-Mour
口試委員:許準榕劉秉慧葉光揚
口試委員(外文):Sheu, Joen-RongLiu, Biing-HuiYeh, Kun-Yun
口試日期:2016-07-20
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生命科學暨生物科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:49
中文關鍵詞:斑馬魚MicroRNA 27bPPARγ脂肪細胞肥胖
外文關鍵詞:zebrafishMicroRNA 27bPPARγadipocyteobesity
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全球目前已有超過19億的成年人列為超重或是肥胖,也造成全球醫療上重要的問題,當脂肪累積過量時,對於健康造成許多負面影響,如糖尿病、心臟病,癌症風險等。而脂肪細胞的形成,主要受到脂肪儲存調節及環境因子所造成的影響,從以往的研究中指出miRNA可以加速或抑制脂肪細胞分化,並且調節其他基因的表現。對於脂肪分化的主要功能是由PPARγ所活化,然而mir27b是在PPARγ非編碼區上的作用位點。Mir27b的損害則會影響到PPARγ及C/EBP的標定物,本研究將透過miRNA-sponge技術,結合具有相同序列中多個miRNA合成,並且針對特異性結合miRNA進行相關的研究。研究中發現在Oil Red O stain時,高油脂餵食mir27b轉基因魚油滴分布較高於wild-type且有明顯差異,另外於RT-PCR半定量分析中,mir27b-sponge轉基因魚能使PPARγ及C/EBP等其脂肪代謝相關的target gene產生上調的趨勢。從H&E染色結果來看,發現在肝臟的部位,能清楚的觀察到mir27b相較於wild-type的脂肪細胞要來的大。結合以上結果得知mir27b確實在脂肪代謝及生成中扮演著重要的角色,因此希望未來能建立與脂肪代謝相關的疾病上能有更進一步的研究。
Obesity has emerged as a global health problem with more than 19 billion adults to be classified as overweight or obese, but also medically important global issue, when excess fat accumulation, and causing negative health effects. If excessive fat accumulation and for many negative health effects like diabetes, heart desease and cancer risk. From previous studies the mir27b may be to PPARγ non-coding regions on the action site, and through the mir27b damage will affect PPARγ and C/EBP calibration. Therefore, we chose MicroRNA-27b as the major research goals through RNA-sponge technology, combined with the same sequence number of miRNA in synthesis, research for specific binding miRNA. From the study found that in mir27b in the Zebrafish, obvious difference for fat build. In theory, mir27b sponge may induce activation of PPARγ, and generate more fat cell differentiation.In Oil o stain the study proved, fatty feeding mir27b sponge transgenic fish oil droplet of distribution is higher than wild type and gives it a significant difference. Beside the RT-PCR to half-quantitative mir27b sponge is able to do transgenic fish the PPARγ and C/EBP be released aim to fat metabolism in connection with trends, mir27b is adipose metabolisms and plays an important role build in him, so that fat metabolisms. Finally, we hope our model fish can provided a platform to study lipid accumulation and early stage adipocyte differentiation.
謝辭 I
摘要 II
Abstract III
目次 IV
圖目次 VI
表目次 VII
壹、緒論 1
(一) 脂肪分化 (Adipose differentiation) 1
(二) 成脂路徑 (Adipogenic pathway) 1
(三) MicroRNA-27b 2
(四) 模式生物 (Model Organisms) – 斑馬魚 2
(五) Tol 2 systems 介紹 3
(六) Beta-actin promoter 4
(七) MicroRNA sponge 4
(八) 研究動機 5
貳、材料與方法 6
(一) 實驗材料 6
1. 生物技術 6
2. 反應試劑 6
3. 儀器 9
(二) 實驗方法 10
1. 生物技術 10
2. Mir27b-sponge Construct 構築 14
3. 基因轉殖魚建立 17
4. 高低能量飼料餵食試驗 18
5. 油紅染色Oil-Red O stain 18
6. 簡易基因表現觀察 18
7. 石蠟切片及H&E染色 20
參、實驗結果 22
(一) βactin-zmCherry-mir27b sponge基因體表現 22
(二) βactin-zmCherry-mir27b sponge轉基因斑馬魚建立 22
(三) Oil red O stain 染色結果 22
(四) mir27 traget gene半定量基因表現 23
(五) H&E染色結果 23
肆、討論 25
伍、參考文獻 27
陸、圖 30
柒、表 48

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