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研究生:陳嘉珮
研究生(外文):Chia-Pei Chen
論文名稱:綠茶表沒食子兒茶素沒食子酸酯藉由微核醣核酸-143/蛋白δ同源物1路徑抑制3T3-L1前脂肪細胞的生長
論文名稱(外文):Green tea epigallocatechin gallate inhibits 3T3-L1 preadipocyte growth through the microRNA-143/DLK1 pathways
指導教授:高永旭
指導教授(外文):Yung-Hsi Kao
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:95
中文關鍵詞:脂肪小分子核糖核酸表沒食子兒茶素沒食子酸酯
外文關鍵詞:AdipocyteMicroRNAEGCG
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據以前報導指出,綠茶兒茶素(GTCs)和小分子核糖核酸(miR)可分別通過改變脂肪細胞的有絲分裂和成脂過程來調節肥胖的發展。但是尚未發現有關綠茶的miR介導效應,特別是表沒食子兒茶素沒食子酸酯(EGCG)對脂肪細胞生長的影響的研究。使用3T3-L1白色前脂肪細胞,我們發現EGCG上調了20個miR分子(例如,mmu-miR-1224-5p,mmu-miR-711和mmu-miR-8108),而下調了20個miR分子(例如mmu-miR -668-3p和mmu-miR-690)。進一步RT-qPCR分析證實,EGCG時間和劑量依賴性地誘導3T3-L1前脂肪細胞miR-143和miR-let-7a水平的增加。且EGCG對miR-155的表達沒有影響,但顯著降低了Pref-1 / DLK1(miR-143的靶標)和HMGA2(miR-let-7a的靶標)mRNA和蛋白質的表現。這些數據顯示EGCG對特定類型miR家族成員具有選擇性作用。此外,在缺少EGCG的情況下,過度表達miR-143或miR-let-7a通過細胞數量的減少和降低細胞活力抑制了3T3-L1前脂肪細胞的生長。而處理miR-143抑制劑可拮抗EGCG對細胞數量和細胞活力的抑制作用。這些數據顯示,EGCG對前脂肪細胞生長具有miR-143依賴性。有趣的是,EGCG類似物穀胱甘肽激活劑,如N-乙醯半胱氨酸,刺激miR-143和miR-let-7a的表現量,而使用穀胱甘肽抑製劑(如丁硫氨酸亞碸亞胺)預處理可阻斷EGCG的作用。此外,發現AMPK抑製劑,如化合物C,可以逆轉EGCG對miR-143和miR-let-7基因表達的影響。總之,EGCG可能通過穀胱甘肽和AMPK的方式激活miR-143和miR-let-7a分子來抑制前脂肪細胞的生長。
Green tea catechins (GTCs) and microRNA (miR) molecules have been reported to regulate development of obesity, respectively, as characterized by changes in the mitogenic and adipogenic processes of fat cells. No studies were found on the miR-mediated effects of GTC, particularly epigallocatechin gallate (EGCG), on fat cell growth. Using 3T3-L1 white preadipocytes, we found that EGCG upregulated 20 miR molecules (e.g., mmu-miR-1224-5p, mmu-miR-711, and mmu-miR-8108) and downregulated 20 miR molecules (e.g., mmu-miR-668-3p and mmu-miR-690). Further RT-qPCR analysis confirmed that EGCG time- and dose-dependently induced increases in miR-143 and miR-let-7a levels in 3T3-L1 preadipocytes. EGCG had no effect on miR-155 expression, but it significantly induced decreases in levels of Pref1/DLK1 (a target of miR-143) and HMGA2 (a target of miR-let-7a) mRNAs and proteins. These data suggest the selective effect of EGCG on particular types of miR family members. In addition, over-expression of either miR-143 or miR-let-7a inhibited 3T3-L1 preadipocyte growth in the absence of EGCG, as indicated by decreased cell number and by reduced cell viability. Whereas, treatment with the inhibitor of miR-143 antagonized the inhibitory effect of EGCG on cell number and cell viability. These data suggest a miR-143-dependent effect of EGCG on preadipocyte growth. Interestingly, EGCG mimicked the glutathione activator, such as N-acetyl-cysteine, to stimulate miR-143 and miR-let-7a, and pretreatment with the glutathione inhibitor, such as buthionine sulphoximine, blocked such EGCG effects. Moreover, an AMPK inhibitor, such as compound C, was found to reverse the effects of EGCG on miR-143 and miR-let-7 gene expressions. In conclusions, EGCG inhibits the growth of preadipocytes through activations of miR-143 and miR-let-7a molecules in glutathione- and AMPK-dependent ways.
目錄
中文摘要 i
Abstract ii
致謝 iii
目錄 iv
表目錄 vi
圖目錄 vi
縮寫與全名對照表 viii
壹、前言 1
一、 肥胖症 1
二、 脂肪細胞 1
三、綠茶 2
四、 綠茶兒茶素與脂肪之關係 2
五、 microRNA小分子核糖核酸 3
六、 microRNA與綠茶兒茶素之關係 4
七、 microRNA與脂肪之關係 4
八、 研究動機與目的 5
貳、材料與方法 7
一、 實驗材料 7
二、 實驗方法 7
1.細胞培養 7
2.藥物處理 7
3.細胞計數方法 8
4.MTT 8
5.轉染作用 8
6.基因分析 9
7.microRNA分析 10
8.西方墨點法 12
9.統計分析 13
參、實驗結果 15
一、EGCG影響前脂肪細胞內miR-143, miR-let-7a及其靶基因的表現 15
二、EGCG影響miR-7052-5p及預測靶基因的表現 16
三、 大量表達miR-143對3T3-L1 細胞數的影響 16
四、 大量表達miR-let-7a對3T3-L1 細胞數的影響 17
五、 大量表達miR-7052-5p對3T3-L1 細胞數的影響 17
六、 miR-143 inhibitor拮抗EGCG對3T3-L1細胞內miR-143, DLK1基因表現及生長的影響 18
七、 miR-7052-5p inhibitor拮抗EGCG對3T3-L1內細胞生長的影響 18
八、miR-143 mimic 和inhibitor影響3T3-L1細胞內蛋白質的表現 19
九、EGCG藉由GSH途徑而影響前脂肪細胞中miR-143、miR-let-7a和靶基因的表現 19
十、EGCG可能藉由AMPK途徑影響前脂肪細胞中miR-143、miR-let-7a和兩者靶基因的表現 21
肆、討論 24
一、 EGCG對3T3-L1前脂肪細胞中miR具有選擇性的影響 24
二、miR-143調節EGCG對3T3-L1細胞的生長的影響 24
三、miR-let-7a調節EGCG對3T3-L1細胞的生長的影響 25
四、miR-7052-5p調節EGCG對3T3-L1細胞的生長的影響 25
五、EGCG通過GSH途徑對前脂肪細胞中miR和基因的影響 26
六、 EGCG通過AMPK途徑對前脂肪細胞中miR、基因和蛋白的影響 27
伍、結論 29
陸、參考文獻 30
柒、附錄 64

表目錄
表一、材料名稱、產品型號及採購來源 37
表二、反轉錄、PCR與qPCR所使用的primers 40
表三、抗體名稱、產品型號及採購來源 41
表四、12% SDS-PAGE之配置 43
圖目錄
圖一、EGCG在不同時間點對3T3-L1前白色脂肪細胞內miR-143, miR-let-7a, miR-155及其標靶基因表現的影響 44
圖二、EGCG隨著處理劑量的不同對3T3-L1前白色脂肪細胞內miR-143, miR-let-7a, miR-155及其標靶基因表現的影響 46
圖三、EGCG對3T3-L1前脂肪細胞的miR-7052-5p, miR-7683-3p及其預測標靶基因表現量的影響 48
圖四、miR-143 mimic對3T3-L1前白色脂肪細胞內miR-143及DLK1基因和細胞增生的影響 49
圖五、miR-let-7a mimic對3T3-L1前白色脂肪細胞增生的影響 51
圖六、miR-7052-5p mimic對3T3-L1前白色脂肪細胞增生的影響 52
圖七、miR-143 inhbitor拮抗EGCG對3T3-L1前白色脂肪細胞內miR-143及其DLK1基因表現和細胞增生的影響 53
圖八、miR-7052-5p inhbitor拮抗EGCG對3T3-L1前白色脂肪細胞細胞增生的影響 55
圖九、miR-143 mimic對3T3-L1前白色脂肪細胞內ERK, AMPK, p62和LC3β蛋白質表現的影響 56
圖十、miR-143 inhibitor對3T3-L1前白色脂肪細胞內ERK, AMPK, p62, LC3β的影響 57
圖十一、EGCG類似Glutathione活化劑NAC不會改變EGCG對3T3-L1前白色脂肪細胞內miR-143, miR-let-7a, DLK1及HMGA2基因表現的影響 58
圖十二、Glutathione抑制劑BSO會拮抗EGCG對3T3-L1前白色脂肪細胞內miR-143, miR-let-7a及DLK1基因表現的影響 59
圖十三、AMPK抑制劑Compound C改變EGCG對3T3-L1前白色脂肪細胞內miR-143, miR-let-7a及DLK1基因表現的影響 60
圖十四、Compound C調節EGCG處理對3T3-L1前白色脂肪細胞內DLK1, ERK , AMPK,p62和LC3β蛋白質表現的影響 61
圖十五、overexpression DLK1拮抗EGCG對3T3-L1生長的影響 63
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