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系統識別號 U0026-1207201911154000
論文名稱(中文) 胞外囊泡在運動抗微膠細胞活化中所扮演的角色
論文名稱(英文) Study the role of extracellular vesicles in exercise-induced anti-microglial activation
校院名稱 成功大學
系所名稱(中) 細胞生物與解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 107
學期 2
出版年 108
研究生(中文) 周鴻霖
研究生(英文) Hong-Lin Chou
學號 T96054010
學位類別 碩士
語文別 中文
論文頁數 47頁
口試委員 指導教授-郭余民
口試委員-吳佳慶
口試委員-鄭宏祺
中文關鍵字 運動  細胞外囊泡  外吐小體  脂多醣 
英文關鍵字 exercise  extracellular vesicles(EVs)  exosome  LPS 
學科別分類
中文摘要 背景知識: 過去的研究顯示,運動具有改善慢性疾病的功能,但詳細的分子機制尚未明確。在運動的諸多好處中,運動的抗發炎功效扮演相當重要的角色。近期的研究顯示,細胞會分泌細胞外囊泡(包括外吐小體、微泡等)進入血液循環,透過血液的流動將訊息傳遞給遠處的細胞。因此,我們假設運動過後所誘導的細胞外囊泡可透過血液循環達到全身性的抗發炎功能。
實驗目的: 測試長期運動訓練後,大鼠血漿分離的外吐小體之抗發炎效果
實驗方法: 七周大的雄性大鼠進行1小時的急性運動及四週的長期運動。在運動及適當休息後,收集其血漿進行奈米粒子追蹤分析以檢測血漿中細胞外囊泡的濃度及大小分布。接著使用離心及聚合物沉澱的方式分離出外吐小體,並利用脂多醣處理的BV2細胞株及脂多醣刺激發炎的小鼠模型來檢測這些外吐小體的抗發炎效果。
實驗結果: 急性(1小時)及長期(四週)的運動皆會促使外吐小體在血液當中的量有顯著提升。免疫染色實驗中,DiI染色的外吐小體可以被BV2細胞株所吸收。而從長期運動及不運動的大鼠血液中抽取的外吐小體在脂多醣誘導發炎的細胞實驗裡,發現預先處理這些外吐小體皆可抑制BV2細胞株中發炎訊號NFκB的表現,其中長期運動的組別抗發炎效果更加顯著。進一步在動物實驗上也發現,長期運動的大鼠抽取出的胞外小體在染色後進行尾靜脈注射,可以藉由血液循環通過血腦障壁,並抑制黑質區中微膠細胞的活化指標Iba1蛋白的表現。
結論: 跑步運動會增加血液循環中的細胞外囊泡,而長期運動大鼠血漿中的胞外小體在運動後抗發炎的效益中扮演相當重要的角色。運動具有改變細胞外囊泡的質與量之功效。
英文摘要 Background: Exercise exerts multiple beneficial effects against chronic diseases, but the detailed molecular mechanism is unclear. It has been suggested that the anti-inflammatory effect contributes to a major part of the exercise benefit. Previous studies have showed that extracellular vesicles (EVs), including exosomes and microvesicles, secreted by cells into circulation playing important roles in long-distance intercellular communication. We hypothesize that the exercise-derived EVs contribute to the exercise-induced anti-inflammatory effects.
Objective: To examine the anti-inflammatory effect of exosomes after long-term exercise training.
Methods: Male SD rats, 7-week old, were subjected to 1-hour and 4-week treadmill running. Their plasma specimens were harvested immediately or 2-day after exercise. The concentration and size distribution of EVs in the plasma were analyzed by Nanoparticles tracking analysis. A combination of centrifugation and polymer-based precipitation method was used to isolate the exosomes. The anti-inflammatory effect of exosomes were examined in vitro using macrophage/microglia cell line, BV2, and in vivo in LPS-challenged mice.
Results: The concentrations of EVs were increased in the plasma of both 1-hour and 4-week exercised rats. Using DiI-labeled exosomes, we showed that exosomes could be absorbed by BV2 cells within 2 hours. Pretreatment of exosomes for 2 hours inhibited LPS-induced activations of NFκB in the BV2 cell, which was more pronounced in the exosomes derived from the 4-week exercise group. Furthermore, 2-hour after tail vein injection, labeled exosomes could be identified in the brain parenchyma. Exosomes derived from rats received 4-week exercise ameliorated the LPS-induced microglial activation in the substantia nigra.
Conclusion: Running exercise increase the concentration of EVs in the plasma. Exosomes derived from long-term exercised rats possess anti-inflammatory effects. Exercise alters the quantity and quality of EVs.
論文目次 Contents
I. Abstract I
II. 中文摘要 III
III. Abbreviation IV
IV. Introduction 1
Benefits of exercise and anti-inflammation 1
Extracellular vesicle 1
Exosomes release and exercise 2
LPS and neural inflammation 3
V. Objective and hypothesis 5
VI. Materials and Methods 6
1. Animals and exercise paradigm 6
1-1 Acute exercise 6
1-2 Long-term exercise 6
1-3 Sub-LT and supra-LT exercise 7
2. Blood lactate level measurement 7
3. Nanoparticle Tracking Analysis (NTA) 7
4. Exosomes isolation 8
5. BV2 micoglial cell culture and treatment 8
6. Exosome uptake 9
6-1 Exosome uptake in vitro (Immunofluorescence) 9
6-2 Exosome uptake in vivo 9
7. Western blotting 10
8. LPS challenge in vivo 11
8-1 Animal model 11
8-2 Immunohistochemistry 12
8-3 Counting cells 12
8-4 Quantifying microglial area 13
9. Statistical analysis 13
VII. Results 14
VIII. Discussion 20
IX. Conclusion 24
X. References 25
XI. Figures 32
Figure 1. The NTA analysis of EVs in plasma from the rats subjected to 1-hour mild-intensity treadmill running. 33
Figure 2. The NTA analysis of EVs in plasma from the rats subjected to 4-week mild-intensity treadmill running. 35
Figure 3. The NTA analysis of EVs in plasma from the rats subjected to sub-LT (15 m/min) and supra-LT (25 m/min) treadmill running. 36
Figure 4. The exosomal marker expression and particle distribution of the exosomes isolated by the modified protocol. 37
Figure 5. The exosome uptake in the murine microglial BV2 cells. 39
Figure 6. The dose-dependent inflammatory signal for NFκB and JNK in 1-hour LPS treated BV2 cells. 40
Figure 7. Activation of NFκB was inhibited by the 4-week exercise-derived exosomes but not the 1-hour exercise-derived exosome in LPS-induced BV2 cells. 41
Figure 8. The exosomes can cross BBB and enter central nerve system. 43
Figure 9. The inflammatory signal expression in different brain regions from the LPS challenged mice. 45
Figure 10. Long-term exercise derived exosomes can mitigate the LPS-induced activations of microglia cells. 46
Figure 11. Model of the exercise-derived exosomes alleviates LPS-induced inflammation in vitro and in vivo. 47
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