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研究生:藍天韻
研究生(外文):Tien-Yun Lan
論文名稱:探討迪皮質醇抑制發炎反應的機制
論文名稱(外文):Characterization of the mechanism of Dexamethasone-mediated anti-inflammatory responses
指導教授:郭津岑
指導教授(外文):Jean-Cheng Kuo
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:44
中文關鍵詞:迪皮質醇腫瘤壞死因子脂多醣
外文關鍵詞:dexamethasoneTNFLPS
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巨噬細胞(RAW264.7細胞)經由脂多醣(LPS)刺激後引起發炎反應,產生的促炎性細胞因子,比如腫瘤壞死因子(TNF-)清除入侵傷口的病源體,但是嚴重的發炎反應導致周圍組織的損害。迪皮質醇(Dexamethasone)是一種糖皮質激素,迪皮質醇與糖皮質激素受體(Glucocorticoid receptor)相互作用來負向調控細胞激素的轉錄;但是,在活化的巨噬細胞中,我們發現迪皮質醇極少影響腫瘤壞死因子的表現,而且並非透過與糖皮質激素受體作用來抑制腫瘤壞死因子的分泌;另外,我們發現迪皮質醇導致腫瘤壞死因子累積在細胞膜的附近。因此,我們假設迪皮質醇透過細胞內運輸系統抑制腫瘤壞死因子的分泌。令人意外地,在活體外,我們發現迪皮質醇促進驅動蛋白(Kinesin)的活性;我們進一步檢驗迪皮質醇對微小管(Microtubule)動態的影響,然後發現,在活體外迪皮質醇不直接影響微小管的合成(Microtubule polymerization),但是在巨噬細胞中,合成速率顯著地成長;我們更進一步證實迪皮質醇對神經營養因子受體(Neurotrophin receptor p75)胞吐作用(Exocytosis)的影響,發現迪皮質醇普遍地提高運輸速率。因此我們回頭檢驗迪皮質醇對於腫瘤壞死因子運輸的影響,發現迪皮質醇不影響腫瘤壞死因子運輸,而且,我們研究活化TLR4後上游訊息的傳遞,發現迪皮質醇降低p38 MAPK訊息傳導。總而言之,我們總結迪皮質醇透過微小管影響胞吐作用。

Macrophages (RAW264.7 cells) activated by lipopolysaccharide (LPS) stimulation induce inflammatory responses. The production of pro-inflammatory cytokines, such as TNF-α, assists with the clearance of invading pathogens at injured sites, but severe inflammation causes damages on the surrounding tissues. Dexamethasone, a glucocorticoid, reacts with glucocorticoid receptor to negatively regulate the transcription of cytokines. However, we found that the treatment of dexamethasone rarely influences LPS-induced TNF-α expression but significantly prohibits its secretion in a glucocorticoid receptor-independent pathway. We also found that dexamethasone treatment causes TNF-α distributed around the plasma membrane. Therefore, we hypothesized that dexamethasone inhibits TNF-α secretion via an intracellular trafficking system. Surprisingly, we found that dexamethasone promotes kinesin activity in vitro. We also found that dexamethasone enhances microtubule dynamics in living RAW264.7 cells, but not directly influence microtubule polymerization in vitro. We further demonstrated the effects of dexamethasone on exocytosis via p75 export analysis, and found that dexamethasone generally enhances trafficking. Thus, we were back to examine TNF-α trafficking pathway, and found dexamethasone does not influence Rab11-mediated TNF-α trafficking. In addition, we investigated the upstream TLR4-dependent signaling transduction, and found that dexamethasone reduces LPS-induced p38 MAPK signaling cascade. To sum up, we concluded that dexamethasone functions in microtubule-mediated exocytosis.
Contents
中文摘要 I
Abstract II
Contents III
Introduction 1
Inflammation 1
LPS-mediated inflammatory responses in macrophages 1
Microtubule-mediated exocytosis in macrophages 2
Dexamethasone-dependent anti-inflammatory responses in macrophages 3
The purpose of research 4
Materials and methods 5
Materials 5
Methods 8
Results 18
Dex suppresses LPS-stimulated TNF-α secretion in activated macrophages via a glucocorticoid receptor-independent pathway 18
Dex does not regulate LPS-induced TNF-α expression 19
Dex treatment causes the accumulation of TNF-α close to cell membrane 19
Dex promotes the kinesin motor activity with lower ATP consumption in vitro 20
Dex promotes microtubule polymerization and depolymerization in vivo 21
Dex generally promotes vesicle trafficking in vivo 22
Dex influences TNF-α trafficking via a Rab11-mediated pathway 22
Dex does not influence TNF-α trafficking via a Munc13-1/Rab37-mediated pathway 23
Dex reduces LPS-induced p38 signaling activation 23
Dex treatment does not influence TNF-α receptor expression on cell surface 24
Discussion 25
Dexamethasone inhibits LPS-induced TNF-α secretion but promotes its trafficking 25
Post-translational modification of microtubule regulates the secretion of cytokines 26
The role of kinesin superfamily members in macrophages 27
References 29
Figures 33
Fig 1. Dex decreases LPS-stimulated TNF-α secretion in activated macrophages via a glucocorticoid receptor-independent pathway. 33
Fig 2. Dex does not regulate LPS-induced TNF-α expression. 34
Fig 3. Dex treatment causes the accumulation of TNF-α. 35
Fig 4. Dex efficiently promotes microtubule motor activity in vitro. 36
Fig 5. Dex promotes microtubule polymerization and depolymerization in vivo. 37
Fig 6. Dex generally promotes trafficking in vivo. 39
Fig 7. Dex influences TNF-α trafficking via a Rab11-mediated pathway. 41
Fig 8. Dex does not influence TNF-α trafficking via a Munc13 /Rab37-mediated pathway. 42
Fig 9. Dex reduces LPS-induced p38 signaling activation. 43
Fig 10. Dex treatment does not influence TNF-α receptor expression on cell surface. 44

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