肥胖是目前全球公認的健康問題。對於肥胖形成的分子機制雖然尚未明瞭，但卻有很多研究皆在肥胖的病人中發現，肥胖往往伴隨著脂肪細胞生理功能的異常以及體內氧化壓力的上升。脂肪細胞除了具有能量儲存的功能之外，目前更發現具有分泌某些生化因子的能力，統稱為Adipokine。其中包括Adiponectin、Leptin、IL-6、TNF-以及PAI-1。多項研究結果顯示，當肥胖引起體內的氧化壓力上升時，會影響脂肪細胞中adipokines的基因表現，造成代謝的異常。粒線體是體內主要產生氧化壓力的場所。在先前本實驗室的研究當中顯示，在受胰島素刺激的脂肪細胞中，GLUT10會大量表現於粒線體中。並且藉由運送L-dehydroascorbic acid (DHA)，也就是維生素C的氧化形式，進入粒線體中，增加細胞對抗氧化逆境的能力。
本實驗主要希望能夠藉由誘導的方式，在生物體外成功模擬出脂肪細胞在肥胖病理下氧化壓力上升的情況，並觀察GLUT10的表現是否對於相關的adipokines基因表現具有影響力。實驗結果發現當我們成功以25mM glucose誘導脂肪細胞中ROS產生時確實會造成GLUT10 以及相關的adipokines基因表現的上升。且在我們以RNAi的形式抑制掉GLUT10的表現後會發現某些adipokines基因的表現確實受到顯著的影響。根據此實驗結果我們推測，GLUT10確實藉由某些機制影響了adipokines的表現，並在氧化逆境下給予脂肪細胞保護力，以維持脂肪細胞正常的生理功能。

Obesity is a worldwide health problem in developed countries for many years. Although the molecular mechanism is still unclear, many researchers indicated that elevated oxidative stress is a major causative factor of obesity. Oxidative stress is primary produced in mitochondria. Excess oxidative stress will affect adipocyte function and further lead to apoptosis of adipocyte. Nowadays, adipocyte not only plays a role in energy store, but also participates in the homeostasis of metabolism by several factor called adipokines. It has been reported that gene expression of adipokines will be affected under oxidative stress.

In the previous finding from our lab indicated that GLUT10 will facilitate transport of L-dehydroascorbic acid (DHA) into mitochondria and further protects the cells against oxidative stress. DHA is the oxidized form of vitamin C and it will be converted with each other to restore the capacity of redox in mitochondria under oxidative stress. .
In this study, we tested three methods to increase oxidative stress in vitro. As a result, we found that 25mM glucose can successfully induce ROS production and elevated expression of several adipokines and GLUT10. In addition, we knockdown/overexpressed GLUT10 to further understanding the relationship between GLUT10 and several adipokines. From these data, we found that gene expression of adiponectin and leptin obviously increased after GLUT10 knockdown. Furthermore, we suggest that GLUT10, as a protector, will cooperate with adiponectin and leptin in some way to against oxidative stress in cell. Moreover, since adiponectin and leptin can decrease the expression of pro-inflammation factors such as IL-6, TNF- and PAI-1, GLUT10 can also prevent further injury of adipocyte and maintain its physiological function under oxidative stress.

摘要...........................................................i
Abstract.....................................................ii
謝誌..........................................................iv
Table of Content...............................................v
List of Tables...............................................vii
List of Figures.............................................viii
Abbreviations..................................................1
Introduction...................................................2
Materials and Methods..........................................8
1. Plasmid................................................8
2. Cell culture...........................................8
3. Cell differentiation...................................8
4. Oil Red O Staining.....................................9
5. Transfection..........................................10
6. Oxidative Stress Induction and Insulin Stimulation....10
7. RNA Isolation and RT-PCR..............................10
8. Primer Design and Quantitative RT-PCR.................11
9. Statistic.............................................12
Results.......................................................13
1. Adipocyte differentiation of 3T3-L1 cells.............13
2. Differential expression of adiponectin, leptin, TNF-α,
PAI-1, IL-6 and GLUT10 between preadipocytes and
adipocytes............................................13
3. Expression level of adiponectin, leptin, TNF-α, IL-6,
PAI-1 and GLUT10 under H2O2 stress (brief exposure)...14
4. Expression level of adiponectin, leptin, TNF-α, IL-6,
PAI-1 and GLUT10 under H2O2 stress producing form
glucose oxidase (sustained exposure)..................15
5. Differential expression of adiponectin, leptin, TNF-α,
IL-6, PAI-1 and GLUT10 under oxidative
stress................................................15
6. Effect of overexpression and RNA interference of GLUT10
on adipokine’s expression by transient assay under
oxidative stress......................................17
7. Construct three mutations on GLUT10 by site direct
mutagenesis...........................................17
Discussion....................................................19
References....................................................19
Tables........................................................49
Figures.......................................................50

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