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系統識別號 U0026-0908201814102000
論文名稱(中文) 探討小分子核糖核酸196a對於亨丁頓舞蹈症之粒線體功能的影響
論文名稱(英文) Investigate the effects of miR196a on mitochondrial functions in Huntington’s disease
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 106
學期 2
出版年 107
研究生(中文) 林佳葳
研究生(英文) Chia-Wei Lin
電子信箱 happy470224@gmail.com
學號 S36054041
學位類別 碩士
語文別 英文
論文頁數 75頁
口試委員 口試委員-姜學誠
指導教授-楊尚訓
口試委員-莊季瑛
口試委員-蔡曜聲
中文關鍵字 亨丁頓舞蹈症  粒線體  小分子核醣核酸196a  Nrf2  HO-1 
英文關鍵字 Huntington’s disease  mitochondria  miRNA-196a  Nrf2  HO-1 
學科別分類
中文摘要 亨丁頓舞蹈症是顯性神經退化性疾病,其成因是在Huntingtin (HTT)基因上有重複複製的CAG(胞嘧啶-腺嘌呤-鳥嘌呤)序列,進而導致過多HTT蛋白質堆積在腦中並造成細胞功能出現異常。在過去的研究中指出,這些異常的HTT蛋白質會去攻擊細胞中的粒腺體進而使粒線體無法清除細胞中的氧化物,最終導致細胞死亡。在我們實驗室之前的研究指出在細胞、基因轉殖小鼠以及從患者身上取得的誘導性多功能幹細胞等亨丁頓舞蹈症模型中,小分子核醣核酸196a能夠減少神經中HTT蛋白質的堆積以及改善小鼠的行為模式。然而目前對於小分子核醣核酸196a在粒線體功能上的影響仍是不清楚的。因此在本篇提出的假說是小分子核醣核酸196a可能透過改善粒腺體的功能來改善亨丁頓舞蹈症所產生的神經退化。在本篇研究中,我們發現在亨定頓舞蹈症的細胞及老鼠模型中有粒線體功能受損以及氧化壓力過多的情況。此外,小分子核醣核酸196a能夠降低細胞中的氧化壓力。為了更深入探討小分子核醣核酸196a是如何減少細胞中的氧化壓力,我們找到了一個與抗氧化壓力有關的蛋白質nuclear factor erythroid 2 related factor 2 (Nrf2),Nrf2在進入細胞核之後會活化下游的抗氧化基因進而使細胞中的氧化壓力下降。然而,當加入小分子核醣核酸196a後卻無法增加Nrf2的表現量。因此,我們又再更進一步去探討小分子核醣核酸196a是否能影響到Nrf2入核,結果中發現到小分子核醣核酸196a能夠增加Nrf2入核並且增加Nrf2下游基因HO-1的表現量。綜合我們的研究結果,我們發現到在亨丁頓舞蹈症中有粒線體功能受損以及細胞氧化壓力增加的情況。此外,小分子核醣核酸196a也許能夠藉由增加抗氧化基因Nrf2入核來增加抗氧化基因HO-1的表現量而使細胞中氧化壓力下降。我們期許這個發現能為亨丁頓舞蹈症提供新的治療策略。
英文摘要 Huntington’s disease (HD) is a dominant, heritable and neurodegenerative disease. It is caused by an expansion of CAG repeats in exon1 of the Huntingtin (HTT) gene. Mutant HTT has been reported to impair mitochondrial functions, causing decrease of mitochondrial membrane potential and ATP production and increase of oxidative stress. In our previous studies, miR-196a provides beneficial effects in HD. However, the protective mechanism of miR-196a on mitochondrial functions is still unknown. We hypothesize miR-196a might promote mitochondrial functions in HD. In this study, we examined the effects of miR-196a on mitochondrial functions in HD striatal cells and HD transgenic mice. The results showed the mitochondrial dysfunction in HD striatal cells and HD transgenic mice. Furthermore, we also found miR-196a could reduce oxidative stress in HD striatal cells. One of antioxidant genes is nuclear factor erythroid 2 related factor 2 (Nrf2), which is related to antioxidant pathway and it is associated with mitochondrial functions. The results indicated the expression level of Nrf2 is decreased in HD striatal cells. Moreover, miR-196a could activate translocation of Nrf2 into nucleus. We also found miR-196a could increase the expression level of the downstream gene, HO-1. In sum, our results suggested mitochondrial dysfunction was found in HD and miR-196a might improve mitochondrial functions through Nrf2 pathway. We anticipate miR-196a should be important for mitochondrial functions, and improved mitochondrial functions may contribute to therapeutic strategies for HD.
論文目次 Abstract I
中文摘要 II
Acknowledgments III
Contents IV
Figure contents VII
Chapter 1. Introduction 1
1.1 Huntington’s disease 1
1.2 Impairment of mitochondrial functions in Huntington’s disease 2
1.3 Oxidative stress in Huntington’s disease 4
1.4 Energy deficit in Huntington’s disease 5
1.5 Nuclear factor erythroid 2 related factor 2 (Nrf2) 5
1.6 Nuclear factor erythroid 2 related factor 2 (Nrf2) in neurodegenerative diseases 7
1.7 The role of Nrf2 in Huntington’s disease 7
1.8 MicroRNA 8
1.9 The role of microRNAs in regulating the Nrf2 pathway 10
Chapter 2. Objectives and specific aims 12
2.1 Research rationales and hypothesis 12
2.2 Specific aims 13
Chapter 3. Materials and methods 14
3.1 DNA construction 14
3.2 DNA transformation 14
3.3 Plasmid DNA extraction 14
3.4 STHdhQ7/Q7 and STHdhQ111/Q111 Cells culture 15
3.5 Transgenic mice 16
3.6 Transfection 16
3.7 Western blotting17
3.8 Immunofluorescence staining 19
3.9 Genotyping for transgenic mice 19
3.10 TMRM staining 20
3.11 Carboxy-H2DFFDA staining 21
3.12 ATP production 22
3.13 Dual Luciferase assay 23
3.14 Fractionation method 23
3.15 Statistical analysis 24
Chapter 4. Results 25
4.1 Mitochondrial membrane potential in vitro and in vivo25
4.2 ATP production in vitro and in vivo 26
4.3 Oxidative stress level in vitro and in vivo 26
4.4 The effects of miR-196a on mitochondrial membrane potential in HD cells 27
4.5 The effects of miR-196a on ATP production in HD cells 28
4.6 The effects of miR-196a on oxidative stress in HD cells 28
4.7 The expression level of antioxidant gene, Nrf2, in vitro and in vivo 29
4.8 The effects of miR-196a on expression level of Nrf2 in HD cells 29
4.9 The effects of miR-196a on translocation of Nrf2 in HD cells 30
4.10 The effects of miR-196a on ARE promoter activity in HD cells 31
4.11 The effects of miR-196a on target genes of Nrf2, HO-1 and GCLC, in HD cells 32
Chapter 5. Discussion 35
5.1 New findings from this thesis 35
5.2 Mitochondrial membrane potential in HD transgenic mice 35
5.3 The effects of miR-196a on mitochondria 36
5.4 The expression level of antioxidant gene, Nrf2, in transgenic mice 37
5.5 The effects of miR-196a on ARE activity 38
5.6 The effects of miR-196a on expression level of GCLC 39
5.7 The working mechanism of miR-196a on oxidative stress 39
5.8The future work 40
Chapter 6. Conclusion 42
Chapter 7. Reference 43
Chapter 8. Figures 50
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