博碩士論文 104821015 詳細資訊




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姓名 張軒嘉(Hsuan Chia Chang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討 PGC-1α與Bhlhe40 的交互作用在生理上所代表的意義
(To investigate the physiological roles of PGC-1α and Bhlhe40 interaction in muscle cells)
檔案 [Endnote RIS 格式]    [Bibtex 格式]    至系統瀏覽論文 (2018-1-25以後開放)
摘要(中) 中文摘要
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)是一個轉錄協同活化因子,調控著氧化代謝,並在調控粒線體生合成以及功能上,扮演極其重要的角色。過去我們實驗室的結果顯示,basic helix-loop-helix family, member e40 (Bhlhe40)與PGC-1α在肌肉細胞中具有很強的結合力,並且Bhlhe40會結合在PGC-1α的啟動子上面抑制其表現量,並且會透過招來HDACs來降低PGC-1α的偕同轉錄活性,另外發現短片段的VP16ADBhlhe40(1-135a.a)-flag (VBHF)可以恢復Bhlhe40對PGC-1α的抑制效果。本篇透過在肌肉細胞中表現短片段的VBHF,探討Bhlhe40與PGC-1α的交互作用在肌肉細胞中所扮演的角色,我們發現,在細胞中表現VBHF後,會降低細胞內的ROS以及粒線體數量,但是卻可以增加細胞的氧氣消耗率,還會增加細胞內過氧化氫小體的數量以及效率,指出Bhlhe40會藉由PGC-1α來影響粒線體以及過氧化小體的功能,進一步的代謝實驗發現,基礎的葡萄糖代謝雖然下降,但是在有胰島素誘導的情況下是上升的,而在脂肪酸代謝方面,粒線體以及過氧化小體方面的脂肪酸代謝都有顯著的上升。除此之外,我們把Bhlhe40的DNA binding domain突變(VBHM)後,發現對於細胞內粒腺體以及過氧化小體的影響消失,代表DNA binding domain對於Bhlhe40去影響粒線體以及過氧化小體的能力是非常重要的。總結來說Bhlhe40會藉由抑制PGC-1α的活性來降低粒線體的功能以及抑制過氧化小體的生合成。最後我們想利用大腸桿菌表現並純化出VBH135,作為一個蛋白質藥,增加肥胖或是代謝不良的病人的代謝效率。
摘要(英) Abstract
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a transcriptional coactivitor participating in the regulation of oxidative metabolism. It is hailed as the master regulator of mitochondria as it coactivates key transcription factors important for mitochondrial function. Our previous results showed that the Bhlhe40 transcription factor repressed the expression of PGC-1α by targeting its proximal promoter and hindered its transactivational ability through recruitment of HDACs. The present study was designed to investigate the physiological roles of PGC-1α and Bhlhe40 interaction in muscle cells. We found that chimeric protein (VBH135) of VP16 activation domain and 1-135 residues of Bhlhe40 rescued the repression effect of wildtype Bhlhe40. Overexpression of VBH135 decreased levels of intracellular ROS and mitochondrion number but increased oxygen consumption, indicating the functional roles of Bhlhe40 on mitochondrial functions. Further metabolic analysis indicated that the basal glucose uptake was decreased but the insulin-induced glucose uptake was increased. Furthermore, mitochondrial and peroxisomal β-oxidation was increased by VBH135. In cells with overexpression of VBH135 the peroxisome number showed a marked increase, implicating the involvement of Bhlhe40 in peroxisomal biogenesis. Increased expression of peroxisome biogenesis-related genes in VBH135 overexpressed cells were identified by microarray assay. Consequently, VBH135 strongly enhanced fatty acids and glucose metabolism. Interestingly, some effects were not found when the DNA binding ability of VBH135 was mutated. Taken together, Bhlhe40 represses mitochondrial function and decreases peroxisome number by negatively regulating PGC-1α at both expression and activity levels. As VBH135 can dominantly rescue wildtype Bhlhe40 repressed oxidative metabolism, it might serve as a metabolic drug for improving the metabolic efficiency in subjects, such as diabetic patients, suffering from low metabolic efficiency.
關鍵字(中) ★ 過氧化小體
★ 粒線體
★ PGC-1α
★ Bhlhe40
關鍵字(英) ★ Peroxisome
★ mitochondria
★ PGC-1α
★ Bhlhe40
論文目次 目錄
一、 緒論 - 1 -
1-1 骨骼肌結構與功能 - 1 -
1-2 粒線體 - 2 -
1-3 過氧化小體 - 5 -
1-4 peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) - 7 -
1-5 basic helix-loop-helix family member e40 (Bhlhe40) - 8 -
1-6研究動機與目的 - 10 -
二、 材料與方法 - 11 -
2-1 細胞株 - 11 -
2-2 RT-PCR - 11 -
2-3 real time PCR mRNA定量實驗 - 13 -
2-4 轉染實驗 - 14 -
2-5 蛋白質標定 - 15 -
2-6 蛋白質純化 - 15 -
2-7 GST pull down assay - 17 -
2-8 ractive oxygen species (ROS)測定 - 17 -
2-9 粒線體膜電位測定 - 18 -
2-10 Genomic DNA 萃取 - 18 -
2-11 葡萄糖代謝測定 - 19 -
2-12 脂肪酸代謝測定 - 19 -
2-13 氧氣消耗測定 - 20 -
2-14 catalase assay - 21 -
2-15 西方墨點實驗 (western blot) - 21 -
2-16 統計檢定方法 - 22 -
三、 實驗結果 - 23 -
3-1短片段Bhlhe40 (1-135a.a)及Bhlhe40 (52-108a.a)會與PGC-1α結合 - 23 -
3-2在C2C12中,建構Tet-off-VP16Bhlhe40 (1-135a.a)-flag (VBHF)及Tet-off-VP16Bhlhe40 (1-135a.a) basic domain mutation-flag (VBHMF) - 23 -
3-3短片段bhlhe40會降低細胞內自由基的合成及使粒線體膜電位下降 - 24 -
3-4 VBHF降低細胞內mtDNA以及數量但是增加粒線體的效率 - 25 -
3-5 VBHF可增加肌肉細胞內過氧化小體的含量,但是在缺少DNA結合能力後,其效果會消失 - 26 -
3-6 VBHF可以增加脂肪酸代謝以及降低基礎葡萄糖攝取,但增加對胰島素所誘導的葡萄糖代謝 - 27 -
3-7、在C2C12中表現VBHF後,利用cRNA micro array觀察細胞mRNA變化,以及影響路徑分析 - 28 -
3-8在C2C12中表現VBHF對於細胞中各個基因mRNA表現量變化 - 29 -
3-9、利用大腸桿菌表現並純化HA2-VP16Bhlhe40 (1-135a.a)-LPRH - 29 -
四、 討論 - 31 -
4-1、細胞內氧化壓力下降,粒線體效率增加 - 31 -
4-2影響過氧化小體生合成以及功能 - 32 -
4-3脂肪酸以及葡萄糖代謝上升 - 33 -
4-4未來發展 - 34 -
4-5代謝性疾病治療 - 34 -
五、 圖表 - 36 -
六、 參考資料 - 51 -
七、 附錄 - 54 -
附錄一:溶液配方 - 54 -
附錄二:引子列表 - 56 -
附錄三:Micro array date - 60 -

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七、
指導教授 陳盛良(Shen-Liang Chen) 審核日期 2017-1-20
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