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研究生:林思廷
研究生(外文):Lin, Szu-Ting
論文名稱:在動物細胞中研究CAP2 和多甲氧基黃酮於粒線體融合及分裂中扮演之角色
論文名稱(外文):Study of the role of CAP2 and Polymethoxylated flavones on mitochondria fusion and fission in mammalian cells
指導教授:張壯榮
指導教授(外文):Chang, Chuang-Rung
口試委員:高茂傑羅至佑兵岳忻
口試日期:2011-7-14
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
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:2011
畢業學年度:99
語文別:中文
論文頁數:60
中文關鍵詞:粒線體甲氧基黃酮天然物融合與分裂
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粒線體參與許多細胞生理代謝的機制,如ATP的生成,鈣離子濃度以及細胞凋亡的調控。粒線體功能不正常時會導致疾病產生。粒線體不斷的進行分裂和融合,除調控粒線體形態並維持正常功能。許多研究發現神經退化性疾病細胞中粒線體形態因動態平衡調控不同而與正常細胞不同。因此在這篇論文中,我從兩個方面來研究影響粒線體動態平衡的機制。在分子機制方面,主要是針對之前實驗室中發現會與粒線體分裂Drp1互相結合的cyclase-associated protein 2(CAP2)。CAP2是一個在演化上高度保留的蛋白,它在調控actin dynamic中扮演重要的角色,也參與Ras/cAMP signaling Pathway。Ras/cAMP signaling Pathway會影響調控Drp1的活性的蛋白激酶A(PKA)。故推測CAP2在動物細胞中會參與粒線體形態調控的機制。我利用轉殖的方式將載體或是RNAi送入在 HeLa cells及SH-SY5Y cells來大量表現或是抑制CAP2,並用共軛焦顯微鏡來觀察粒線體形態的改變。實驗結果證實CAP2會影響融合與分裂的平衡而導致片段狀或網狀的粒線體。在論文第二部分則是探討天然藥材成份對粒線體形態調控的影響。中藥藥材中使用的乾燥柑橘屬果皮中常見多甲氧基黃酮(PMF),尤其是川陳皮素(Nobiletin)及橘皮素(Tangeretin)。目前已知多甲氧基黃酮能抗發炎,抗癌症以及抗粥狀動脈硬化。研究發現也發現多甲氧基黃酮以及5號碳去甲氧基黃酮會參與癌細胞的細胞凋亡,粒線體動態平衡調控與細胞凋亡有密切關聯。為探討多甲氧基黃酮以及5號碳去甲氧基黃酮對於粒線體之影響,我分別使用不同濃度的川陳皮素、橘皮素和5號碳去甲氧基的川陳皮素、橘皮素來檢測其對HeLa cell的細胞存活率以及粒線體形態的影響。實驗結果發現PMFs濃度越高細胞存活率越低,且在10 μM濃度下會影響粒腺體的動態平衡。這些橘皮萃取物具有治療因粒線體導致神經元退化疾病的天然藥物之潛力。

Mitochondria are dynamic organelles that involved in ATP synthesis, calcium concentration regulations and apoptosis. Aberrant mitochondrial morphology caused by abnormal equivalence of fusion and fission in were found in neurodegenerative patients. The aim of my thesis is to study the regulation of mitochondria dynamics. In the first part, I focused on the protein that interacts with mitochondria fission Drp1 called CAP2 (cyclase associated protein 2). CAP2 is a highly conserved protein that plays critical roles in regulating acting dynamics and Ras/cAMP signaling pathway. I manipulated CAP2 expression levels by transfecting either overexpression vectors or RNAi and applied confocal microscope to monitor mitochondrial morphology. My experiments found that overexpression of CAP2 caused mitochondria fragmentation and inhibition of CAP2 lead to elongation. These results indicated that CAP2 does involved in regulating mitochondria dynamics. The second part of my thesis is focus on the effects of nature products on mitochondria dynamics. The dehydrated citrus peels used in traditional Chinese medicine contain the most common flavones: polymethoxylated flavones (PMFs). Both nobiletin and tangeretin are abundant PMFs in citrus peels. Many researches had demonstrated that PMF and 5-OH PMFs involved in anti-inflammatory, antioxidants, antithrombotic and anticarcinogenic processes and affected apoptosis. In my experiments, the survival rates of Hela cells treated with different concentrations of nobiletin, tangeretin, 5-OH nobiletin and 5-OH tangeretin were dosage dependent. In addition, 10 μM PMFs treatments altered mitochondrial dynsmics in HeLa cells. Our results of PMFs suggested these molecules may be therapeutically useful in treating the neurodegenerative disorders caused by mitochondrial fusion and fission regulation defects.
CONTENTS
中文摘要 ii
ABSTRACT iii
誌謝 v
CONTENTS vii
LIST OF FIGURES ix
LIST OF TABLES xi
ABBREVIATION xiii
Chapter 1 General introduction 1
1.1 Structures and functions of mitochondria 1
1.2 Mitochondrion and neurodegenerative diseases 2
1.3 Mitochondrial dynamics, function and diseases 3
1.4 Goal of this study 4
Chapter 2 Material and methods 6
2.1 Plasmids extraction 6
2.2 Cells and Cell culture 6
2.3 Cell lines Transfection 7
2.4 Antibody purification 7
2.5 SDS PAGE and Western blotting 8
2.6 Drug resistance assay and cell viability assay 9
2.7 Fixation 9
2.8 Immunofluorescent Cell Staining 9
2.9 Statistics 9
Chapter 3 The role of CAP2 in mitochondria fusion and fission regulation. 11
3.1 Introduction 11
3.2 Results 13
3.2.1 Verifications of antibodies that can recognize CAP2 13
3.2.2 Overexpression CAP2 in SH-SY5Y cells causes mitochondrial elongation…………………………………………………………… 15
3.2.3 Knockdown CAP2 by RNAi in CAP2-overexpressed in SH-SY5Y cells causes mitochondrial fragmentation………………………………… 16
3.2.4 Overexpression CAP2 in HeLa cells caused mitochondria elongation. …………………………………………………………...17
3.2.5 Knockdown CAP2 by RNAi in CAP2-overexpressed HeLa cells caused mitochondrial fragmentation…………………………………………18
3.2.6 Discussion……………………………………………………………...20
Chapter 4 The role of Polymethoxylated flavones (PMFs) on mitochondrial
fusion and fission regulation..………………………………………23
4.1 Introduction 23
4.2 Results 25
4.2.1 Survival of the PMF treatment effect is dosage dependent……………25
4.2.2 Mitochondrial morphology in Hela cells is affected after PMFs and
5-OH PMFstreatments………………………………………………….26
4.2.3 Discussion…………………………………………………………….27
REFERENCE 29


LIST OF FIGURES
Figure 1 Mitochondria dynamic is regulated by fission and fusion processes. 35
Figure 2 cAMP pathway regulates PKA activity to phosphorylate Drp1 at Ser637 and downregulate the GTPase activity in mammalian cells. 36
Figure 3 Srv2/CAP function domains 37
Figure 4 Ras/cAMP/ adenyl cyclase pathway in yeast. 38
Figure5 Evaluating CAP2 antibody, using 3809 #3 and 3809 #4 serum in western blot with SH-SY5Y total cell lysate. 39
Figure 6 Reduing serum vo;ume and increasing Tween 20 to improve Western blot background. 40
Figure 7 Diagram of antibody purification 41
Figure 8 Different fraction samples taken out during antibody purification on
SDS-PAGE. 42
Figure 9 Evaluation of purified antibody. 43
Figure 10 Evaluating purified antibodies by Odyssey® Infrared Imaging system. 44
Figure 11 Evaluating 3810 #3 and purified 3810 #3 for Western blotting. 45
Figure 12 Using anti-c Myc for detecting CAP2 in overexpressed cells 46
Figure 13 Anti yeast C-terminal Srv2 can be used to detect CAP2 in mammalian cells 47
Figure 14 CAP2 overexpress in SH-SY5Y cells 48
Figure15 Alteration of CAP2 expression affects mitochondrial morphology in SH-SY5Y cells. 49
Figure 16 Identifying optimized ratio of Lipofectamine™ 2000 to CAP2 RNAi by Western blot for SH-SY5Y cells. 50
Figure 17 CAP2 RNAi can inhibit overexpressed CAP2 in SH-SY5Y cells 51
Figure 18 CAP2 RNAi can inhibit overexpressed CAP2 in HeLa cells. 52
Figure 19 CAP2 expression levels affects mitochondrial morphology
in HeLa cells. 53
Figure 20 The structure of PMFs and 5-OH PMFs. 54
Figure 21 Monitoring cell survival after treated with PMFs by MTT assay 55
Figure 22 PMFs and 5-OH PMFs treated cells affect mitochondrial morphology in 10 μM.. 56
Figure 23 Quantitative data of classifications demonstrated that 10 μM PMFs and 5-OH PMFs treated cells affect mitochondrial morphology. 57

LIST OF TABLES

Table 1 Transfection programs. 58
Table 2 Antibodies used in Western blot 59
Table 3 The CAP protein peptide as antigen to product the specific antibody 60














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