臺灣博碩士論文加值系統

骨關節炎(osteoarthritis; OA)是關節炎的最常見形式，而且影響全世界大部分的老年人口，但是OA的發病機制仍然沒有完全理解，因為涉及遺傳、生化和機械等複雜的相互作用。抵抗素 (resistin) 是一種二聚體蛋白，主要是在小鼠中明顯誘導胰島素抵抗而獲得其名稱。先前文獻報導在人類發炎時的狀態，單核球趨化蛋白-1 (MCP-1/CCL2) 是關鍵的趨化因子，其能夠幫助調節單核球細胞的移動和浸潤至發炎區域。而在退化性關節炎的發病機制中常常可以看到單核球細胞的移動和浸潤現象產生在滑液囊發炎區域，促進發炎反應並且加劇退化性關節炎的病症。然而，抵抗素對骨關節炎滑膜成纖維細胞（OASF）的影響仍然未知。在我們的研究中發現，抵抗素被發現激活磷脂酰肌醇-3-激酶（PI3K），Akt和哺乳動物雷帕黴素靶蛋白（mTOR）信號通路，而PI3K，Akt和mTOR抑製劑或小干擾RNA減少抵抗素誘導的MCP-1表達和單核細胞遷移。此外，通過PI3K，Akt和mTOR信號傳導途徑抑制miR-33a和miR-33b，抵抗素被證明可刺激MCP-1介導的單核細胞遷移。這些結果提供了抵抗素機制的新見解，並可能對OA患者俱有治療意義。
此外，許多證據證明白細胞介素-1β(Interleukin 1 beta; IL-1β)、腫瘤壞死因子α（Tumor Necrosis Factor-α; TNFa）是參與OA的病理生理學的主要促炎細胞因子。在退化性關節炎軟骨細胞中，IL-1β以及TNFa表現顯著增加，不僅抑制細胞外基質的合成，還可以刺激基質金屬酵素蛋白酶的釋放。然而，抵抗素是否在退化性關節炎滑液囊纖維細胞 (Osteoarthritis synovial fibroblast; OASF)中調控IL-1β以及TNFa的表現仍然未知。我們的研究結果發現，抵抗素可增加退化性關節炎滑液囊纖維細胞中的IL-1β以及TNFa表現。抵抗素可在退化性關節炎滑液囊纖維細胞誘導MEK和ERK (Extracellular signal-regulated kinases)磷酸化。抵抗素誘導的IL-1β以及TNFa表現會被MEK和ERK抑製劑所拮抗而更進ㄧ步證實訊息路徑參與。此外，抵抗素增強IL-1β以及TNFa表達涉及MEK和ERK信號傳導途徑，而miR-149表達受到抵抗素通過MEK和ERK途徑的負面影響。我們的結果說明了退化性關節炎的臨床意義並且探討對於退化性關節炎滑液囊纖維細胞訊號傳遞機制的了解，期望可以帶來更多的降低發炎反應的治療新策略。

Osteoarthritis (OA) is the most common form of arthritis and affects a large portion of aged population worldwide. The pathogenesis of osteoarthritis (OA) is still not fully understood due to the complex interaction of genetic, biochemical, and mechanical factors involved in this disease. Resistin is a dimeric protein that may be involved in the pathogenesis of multiple inflammatory diseases including osteoarthritis. Have been reported that MCP-1 play a critical role in the pathogenesis of OA by regulating monocyte migration and infiltration. However, the effects of resistin on monocyte chemoattractant protein-1 (MCP-1) expression in osteoarthritis synovial fibroblasts (OASFs) remain unknown. In this study, resistin was found to activate the phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) signaling pathways, while PI3K, Akt, and mTOR inhibitors or small interfering RNAs diminished resistin-induced MCP-1 expression and monocyte migration. In addition, resistin was shown to stimulate MCP-1-mediated monocyte migration by suppressing miR-33a and miR-33b via the PI3K, Akt and mTOR signaling pathways. These results provide new insights into the mechanisms of resistin and may have therapeutic implications for patients with OA.

In addition, much evidence has revealed that interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNFa) are the main proinflammatory cytokine involved in the pathophysiology of OA. In OA chondrocytes, levels of IL-1β and TNFa are markedly increased, which not only suppresses the synthesis of the extracellular matrix (ECM), but also stimulates the release of catabolic proteases such as matrix metalloproteinase (MMPs). However, the effects of resistin on IL-1β and TNFa responses in OA remain unknown. Here, our preliminary data showed that pretreatment of cells with resistin increased IL-1β and TNFa expression in OA cells. Our data showed that resistin induced MEK, and ERK phosphorylation. In addition, resistin-induced IL-1β and TNFa expression were antagonized by MEK, and ERK inhibitors. Therefore resistin enhances IL-1β and TNFa expression involved in MEK and ERK signaling pathways, whereas miR-149 expression is negatively affected by resistin via the MEK and ERK pathways. Our results illustrate the clinical significance of degenerative OA and explore the understanding of the signaling mechanisms of the synovial fibroblasts, which are expected to lead to new therapeutic strategies to reduce inflammatory responses.
Osteoarthritis (OA) is the most common form of arthritis and affects a large portion of aged population worldwide. The pathogenesis of osteoarthritis (OA) is still not fully understood due to the complex interaction of genetic, biochemical, and mechanical factors involved in this disease. Resistin is a dimeric protein that may be involved in the pathogenesis of multiple inflammatory diseases including osteoarthritis. Have been reported that MCP-1 play a critical role in the pathogenesis of OA by regulating monocyte migration and infiltration. However, the effects of resistin on monocyte chemoattractant protein-1 (MCP-1) expression in osteoarthritis synovial fibroblasts (OASFs) remain unknown. In this study, resistin was found to activate the phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) signaling pathways, while PI3K, Akt, and mTOR inhibitors or small interfering RNAs diminished resistin-induced MCP-1 expression and monocyte migration. In addition, resistin was shown to stimulate MCP-1-mediated monocyte migration by suppressing miR-33a and miR-33b via the PI3K, Akt and mTOR signaling pathways. These results provide new insights into the mechanisms of resistin and may have therapeutic implications for patients with OA.

In addition, much evidence has revealed that interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNFa) are the main proinflammatory cytokine involved in the pathophysiology of OA. In OA chondrocytes, levels of IL-1β and TNFa are markedly increased, which not only suppresses the synthesis of the extracellular matrix (ECM), but also stimulates the release of catabolic proteases such as matrix metalloproteinase (MMPs). However, the effects of resistin on IL-1β and TNFa responses in OA remain unknown. Here, our preliminary data showed that pretreatment of cells with resistin increased IL-1β and TNFa expression in OA cells. Our data showed that resistin induced MEK, and ERK phosphorylation. In addition, resistin-induced IL-1β and TNFa expression were antagonized by MEK, and ERK inhibitors. Therefore resistin enhances IL-1β and TNFa expression involved in MEK and ERK signaling pathways, whereas miR-149 expression is negatively affected by resistin via the MEK and ERK pathways. Our results illustrate the clinical significance of degenerative OA and explore the understanding of the signaling mechanisms of the synovial fibroblasts, which are expected to lead to new therapeutic strategies to reduce inflammatory responses.

中文摘要 i
English Abstract iii
Acknowledgement……………………………………………………………………….v
目錄 vi
List of Tables viii
List of Figures ix
Abbreviations xi
Chapter I: General Introduction 1
1.1 Introduction 1
1.2 Phenotypes of osteoarthritis 3
1. Post traumatic 4
2. Aging 5
3. Genetics 7
4. Metabolic 7
1.3 The role of resistin in osteoarthritis 9
1.4 The role of monocyte chemoattractant protein-1 (MCP-1) in osteoarthritis 12
1.5 The role of interleukin-1β (IL-1β) in OA 16
1.6 MicroRNAs (miRNAs) in osteoarthritis 19
Chapter II: Materials and Methods 22
2.1 Materials 22
2.2 Human synovial tissues 23
2.3 Cell culture 23
2.4 Western blot analysis 24
2.5 Quantitative real-time polymerase chain reaction 25
2.6 In vitro chemotaxis assay 25
2.7 ELISA assay 26
2.8 Cell transient transfection 26
2.9 Statistical analysis 27
Chapter III: Resistin Enhances Monocyte Chemoattractant Protein-1 Production and Facilitates Monocyte Migration in Human Synovial Fibroblasts 28
3.1 Results 28
3.1.1 Resistin promotes MCP-1 production in human synovial fibroblasts and facilitates monocyte migration 28
3.1.2 The PI3K/Akt signaling pathway plays a role in resistin-induced MCP-1 expression and monocyte migration 30
3.1.3 Resistin increases MCP-1 production and facilitates monocyte migration via the mTOR pathway 33
3.1.4 Resistin facilitates MCP-1-related monocyte migration by suppressing miR-33a/miR-33b 35
3.2 Discussion 39
Chapter IV: Resistin Activates IL-1β in Human Synovial Fibroblasts 43
4.1 Results 43
1. Resistin mediated TNFα and IL-1β expression in OASFs 43
4.1.2 The MEK/ERK signaling pathway plays a role in resistin-induced TNF-α and IL-1β expression 45
4.1.3 Resistin facilitates TNF-α and IL-1β expression by suppressing miR-149 48
Chapter V: Conclusion 53
References 55

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