||Exercise in an Electrotatic Flow Chamber Ameliorates Age-Related Degeneration in Caenorhabditis elegans
||Department of BioMedical Engineering
Caenorhabditis (C.) elegans
運動已被證實能夠讓身體變得更健康並且降低多種疾病的發生率。近期的研究指出，規律的運動可以提升腦部的保護力，並且降低多種疾病的早期發病率。脂褐素是一種在細胞內累積的代謝廢物，在許多研究中認為脂褐素對動物而言是有害的。然而，在我們的研究中發現，與其說脂褐素對人類有立即的威脅性，將其稱之為老化的指標物更加的貼切。先前的研究指出，脂褐素可作為阿茲海默症發病程度的指標。在我們以前的研究中，表現Aβ蛋白而行動能力下降的線蟲，能透過適度且規律的運動而提升行動能力。適度且規律的運動能夠提升體內的抗氧化物含量，包括超氧化物歧化酶(SOD)、過氧化氫酶(CAT)以及穀胱甘肽過氧化物酶(GPx)。因此本研究假設適度的運動能藉由提升抗氧化物，促進細胞內清除有害物質的能力，並且及早避免退化性疾病的發生。本篇研究中，探討運動帶來的效益會不會由於缺少如CAT或SOD這樣重要的抗氧化物而遺失。為了簡化研究，本研究所使用的模板生物為學名秀麗隱桿線蟲的無脊椎多細胞動物。由於這種線蟲是最小的多細胞動物，構造簡單，生命週期短暫，所有基因組已被完全定序而且有60%的基因與人類相同，因此成為本計畫理想的研究對象。我們以N2為野生株，另外分別以基因變異株LB90與GA480線蟲做為CAT與SOD缺陷的代表。論文中，利用線蟲的趨電性誘導線蟲在趨電晶片上來回游動。透過ELISA分析與一個利用微觀粒子影像流速儀(micro particle image velocimetry)取得加速度場的圖像基礎演算法，運動訓練結束後六小時的線蟲可進行氧化壓力與行動能力的分析。除此之外，還有進行老化指標(脂褐素堆積)、子代數目與壽命等評估，藉以比較運動訓練對線蟲生理狀態所產生的影響。在本研究中發現，CAT與SOD對於降低氧化壓力而言是必要的，其中SOD的重要性更勝CAT一籌。然而，除了這兩者之外仍存在著其他降低氧化壓力的途徑。最後，運動能促使肌肉成長得更強壯，並且連帶使子代數目增加。
Physical exercise has been proven an effective measure to enhance wellness and prevent diseases. The recent literature has shown that regular exercise is able to boost brain protection and reduce the early onset of several diseases. Lipofuscin (LF) is an aggregation of metabolic leftover that is regarded harmful to animals in many studies, whereas in our research, LF was found to be more like an indicator rather than an immediate threat to humans. Prior studies have indicated that LF can be an indicator associated with the degree of Alzheimer's disease (AD). A previous research suggested that exercise training can not only reduce the LF accumulation but also restore the motility of AD worms (CL2120 strain). It has been well known that moderate and regular exercise can upregulate antioxidants, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Therefore, it is assumed that appropriate exercise will scavenge harmful substances in cells by escalating antioxidants level in the early stage and then prevent the degenerative diseases. In this study, it is investigated whether the deficiency of related antioxidants such as SOD and CAT will erase the good effect of exercise. To simplify the intricate research, the model animal chosen in this study is Caenorhabditis elegans (C. elegans), a simple round worm with more than 60% genetic similarity with humans. The N2 strain was used as wide-type worm, and transgenic strains LB90 and GA480 were used as CAT-deficit and SOD-deficit models respectively. In this thesis, nematodes were induced to swim back and forth on an electrotactic microchip. Using an ELISA method and a self-developed image-based algorithm that can obtain the acceleration fields by micro particle image velocimetry, the worms underwent the evaluation of oxidative stress and kinetic power 6 hours after the exercise treatment. The lifespan, progeny and age pigment (lipofuscin) of the nematodes were also analyzed in order to compare the physiological changes brought by exercise treatment. In our findings, CAT and SOD enzymes are necessary to reduce ROS, and among them SOD appeared to be more important than CAT. However, it was indicated that there still exists other ROS-scavenging pathways. Lastly, exercise can improve muscle strength and boost the motility as well as progeny in nematodes.
LIST OF FIGURES IX
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 Caenorhabditis (C.) elegans 2
1.3 Oxidative Stress 3
1.4 Antioxidants 4
1.5 Lipofuscin(LF) 6
1.6 Aims of the Thesis 8
CHAPTER 2 MATERIALS AND METHODS 10
2.1 Worm Strains and Culture Conditions 10
2.2 Electrotactic Microchip (Worm Treadmill) 11
2.2.1 Electrotactic Microchip Fabrication 11
2.2.2 Exercise treatment operation 12
2.3 Immobilization Method 12
2.4 Assessment of Mitochondrial Diameter 13
2.5 Lifespan Assay 13
2.6 Progeny Assay 14
2.7 Lipofuscin Accumulation Analysis 14
2.7.1 Nile Red Staining 14
2.7.2 Quantification of Lipofuscin Accumulation 15
2.8 Measurement of Reactive Oxygen Species 15
2.9 Motility Assessment 16
2.9.1 Derivation of Kinetic Power 16
2.9.2 Measurement Methods 17
CHAPTER 3 RESULTS AND DISCUSSIONS 19
3.1 Effect of Electric Field 19
3.2 Mitochondrial Diameter 21
3.3 Lipofuscin Accumulation 27
3.3.1 LB90 27
3.3.2 GA480 29
3.4 Lifespan Assay 31
3.4.1 LB90 31
3.4.2 GA480 32
3.5 Progeny Assay 33
3.6 ROS Assay 36
3.6.1 N2 36
3.6.2 LB90 37
3.6.3 GA480 39
3.7 Kinetic Power Assay 41
3.7.1 LB90 41
3.7.2 GA480 42
CHAPTER 4 CONCLUSION 45
CHAPTER 5 FUTURE WORK 48
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