臺灣博碩士論文加值系統

過去已有許多的研究指出，粒線體在幹細胞分化及成熟細胞中扮演了重要的角色。另外，粒線體及脂肪細胞功能的異常也被證實與第二型糖尿病有著密切的關係，然而目前對於粒線體功能異常導致第二型糖尿病的機制仍不清楚。在本研究中，我主要探討粒線體中之 Sirtuin 3 蛋白在衍生自人類脂肪組織之間葉幹細胞進行脂肪細胞分化及抗氧化防禦調控機制所扮演的角色。我發現在脂肪細胞分化的過程中，粒線體生合成相關之基因與呼吸酵素次單位蛋白之表現量皆有顯著上升，而粒線體中的活性氧分子與粒線體之膜電位也在分化刺激後短時間內有顯著的增加，抗氧化酵素之mRNA與蛋白表現量也在細胞分化過程中有顯著的上升。此外，我發現粒線體中之 Sirtuin 3 蛋白表現量在脂肪細胞分化的過程中有顯著的增加。為了進一步探討 Sirtuin 3 在脂肪細胞分化過程中所扮演的角色，我先利用病毒感染人類間葉幹細胞來降低其蛋白表現量，再使其進行脂肪細胞分化。我首先分析在 Sirtuin 3 knockdown 的脂肪細胞中，其脂肪分化及與脂肪細胞功能相關基因的表現量，結果顯示 Sirtuin 3 knockdown 會造成這些基因的表現下降。進一步分析粒線體生合成之相關基因的表現，我發現 Sirtuin 3 knockdown 的脂肪細胞有顯著受到抑制的現象。此外，我觀察到 Sirtuin 3 knockdown 的人類間葉幹細胞分化而成之脂肪細胞的粒線體膜電位有顯著的下降，而粒線體中活性氧分子則呈顯著的上升。另一方面，我探討細胞中抗氧化酵素基因之表現，發現 Sirtuin 3 knockdown 會導致這些基因的表現受到抑制。綜合以上的實驗結果，我證明在衍生自脂肪組織之人類間葉幹細胞分化為成熟脂肪細胞的過程中，Sirtuin 3 的蛋白表現與粒線體生合成及呼吸作用有著密切的關係。我也進一步闡明 Sirtuin 3 的表現量下降會造成脂肪細胞之粒線體功能異常，以及抗氧化防禦機制受損，並增加細胞中之氧化壓力，這些損傷可能是 Sirtuin 3 缺少導致脂肪細胞分化受到抑制的原因。

Mitochondria play important roles in the differentiation and functioning of stem cells. Abundant evidence has shown that mitochondrial dysfunction in adipocytes is associated with type 2 diabetes. However, the mechanism by which mitochondrial dysfunction causes type 2 diabetes remains to be elucidated. In this study, the roles that sirtuin 3 (SIRT3), a mitochondrial protein, plays in adipogenic differentiation, mitochondrial biogenesis and antioxidant defense system of adipocytes differentiated from human adipose tissue-derived mesenchymal stem cells (ad-hMSCs) were investigated. The results showed that the mRNA levels of mitochondrial biogenesis-related genes, peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1), and the protein levels of respiratory enzyme complex subunits were up-regulated during adipogenic differentiation of ad-hMSCs. The levels of mitochondrial membrane potential and reactive oxygen species (ROS) in mitochondria were increased, and the mRNA and protein levels of the antioxidant enzymes Mn-SOD and catalase were up-regulated during adipogenic differentiation of ad-hMSCs. In addition, the protein level of SIRT3 was dramatically increased during adipogenic differentiation of ad-hMSCs. To further investigate the role of SIRT3 in adipogenic differentiation, I infected ad-hMSCs with lentivirus to suppress the protein expression of SIRT3 and examined its impact on adipogenic differentiation. The protein levels of adipogenic markers, including CCAAT enhancer binding protein  (C/EBP), peroxisome proliferator-activated receptor  (PPAR), fatty acid binding protein 4 (FABP4) and adiponectin were decreased during adipogenic differentiation of ad-hMSCs with SIRT3-knockdown. In addition, the mRNA and protein levels of PGC-1and mitochondrial membrane potential were decreased by SIRT3 knockdown during adipogenic differentiation of ad-hMSCs. The mitochondrial ROS levels were increased and the protein levels of antioxidant enzymes were decreased by SIRT3 knockdown during adipogenic differentiation of ad-hMSCs. These findings together suggest that SIRT3 plays important roles in mitochondrial biogenesis, adipogenic differentiation and the antioxidant defense of adipocytes. Moreover, I demonstrated that SIRT3 deficiency caused an increase of the oxidative stress level in mitochondria and resulted in mitochondrial dysfunction, which in turn impaired the adipogenic differentiation of ad-hMSCs.

中文摘要 .................................. 3
Abstract ................................. 4
Abbreviations ............................ 6
Introduction
I. Stem Cells ......................... 8
II. Mitochondria ....................... 11
III. Sirtuins .......................... 15
IV. Diabetes Mellitus .................. 18
Rationale and Significance ............... 20
Materials and Methods .................... 21
Results .................................. 31
Discussion ............................... 39
References ............................... 46
Figures .................................. 58
Figure 1. Morphological changes during adipogenic differentiation of ad-hMSCs .............. 58
Figure 2. Increase of the expression of adipogenic
markers during adipogenic differentiation of ad-hMSCs
.......................................... 59
Figure 3. Increase in the expression of the subunits of respiratory enzyme complexes during adipogenic differentiation of ad-hMSCs .............. 60
Figure 4. Increase of mitochondrial membrane potential (m) at the early stage of adipogenic differentiation of ad-hMSCs .......................................... 61
Figure 5. Dramatic increase of the mRNA levels of genes related to mitochondrial biogenesis during adipogenic differentiation of ad-hMSCs .............. 62
Figure 6. Increase of mitochondrial reactive oxygen species during adipogenic Differentiation of ad-hMSCs
.......................................... 63
Figure 7. Increase in the expression of antioxidant enzymes during adipogenic differentiation of ad-hMSCs .......................................... 64
Figure 8. Increase in the protein expression of SIRT3 during adipogenic differentiation of ad-hMSCs ... 66
Figure 9. Decrease of the induction fold of SIRT3 after lentiviral infection ..................... 67
Figure 10. Inhibition of lipid droplets formation in adipocytes differentiated from SIRT3-knockdown ad-hMSCs .......................................... 68
Figure 11. Decrease in the mRNA expression of adipogenic marker genes in adipocytes differentiated from SIRT3-knockdown ad-hMSCs ....................... 69
Figure 12. Decrease in the expression of PGC-1 in adipocytes differentiated from SIRT3-knockdown ad-hMSCs .......................................... 71
Figure 13. Decrease of the mitochondrial membrane potential in ad-hMSCs with SIRT3-knockdown at the early stage of adipogenic differentiation ............... 72
Figure 14. Increase of mitochondrial ROS in SIRT3-knockdown ad-hMSCs at different time points after adipogenic differentiation .......................... 73
Figure 15. Decrease of the mRNA expression levels of antioxidant enzymes during adipogenic differentiation of ad-hMSCs with SIRT3-knockdown ............... 74
Figure 16. A schematic summary of the results obtained in this study ............................... 75

Tables ................................... 76
Table 1. Oligonucleotide sequences of primers and Taqman probes used in this study ................ 76
Table 2. Antibodies used in this study ... 77

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