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研究生:李家銘
研究生(外文):Jia-ming Li
論文名稱:探討運動促進造血前驅細胞的動員對於周邊神經壓傷修復之影響
論文名稱(外文):Exercise promotes mobilization of hematopoietic progenitor cells involved in repair of sciatic nerve crush injury
指導教授:王銘富王銘富引用關係
指導教授(外文):Ming-fu Wang
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:85
中文關鍵詞:神經再生輪帶跑步機運動造血前驅細胞坐骨神經壓迫
外文關鍵詞:treadmill exercisesciatic nerve crush injurynerve regenerationhematopoietic progenitor cells
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造血前驅細胞(Hematopoietic progenitor cells)影響周邊神經再生之過程,大部分是經由CD34+細胞的聚集而產生效應。輪帶跑步機運動(treadmill exercise)在各種不同強度或頻次之下,可增加軸突再生與神經分支,但是輪帶跑步機運動後對於骨髓細胞的動員而參與神經修復的可能性,目前研究的結果還尚未定論。
使用血管夾夾在Sprague-Dawley大白鼠的左側坐骨神經進行夾傷實驗,來誘發周邊神經損傷。將動物分成兩組,分別是crush group (未做運動)與輪帶跑步機運動組(Crush+ Treadmill)(每天跑20米/分鐘,持續一小時),實驗為期壹週。使用Cytospin和細胞流式儀來測量骨髓細胞的分佈及密度。神經功能、神經電生理及神經再生的參數於運動後的第一及三週實行。使用免疫組織染色及西方墨點的方法來測量造血前驅細胞、免疫細胞及血管生成因子的表現和分布。
輪帶跑步機運動具有顯著促進神經再生的功能。輪帶跑步機運動組在實驗7天後,在神經組織中會顯著增加許旺氏(Schwann)細胞的增殖、降低許旺氏細胞細胞凋亡(apoptosis) 以及增進神經纖維(neurofilament)大量的表現。夾傷之後的神經,在運動過後不但提升神經鞘化的程度﹝如S-100與Luxol Fast Blue的表現增加﹞而且降低神經組織的空泡數目。輪帶跑步機運動後會誘導骨髓細胞顯著增加其單核球細胞數目,尤其是CD34+細胞。運動之後會增加神經組織中CD34+細胞的堆積的數目及表現,而此現象會導致Von-Willebrand, Isolectin B4和VEGF的血管生成因子的同步上升。
經由低強度的輪帶跑步機運動可刺激骨髓中造血前驅細胞特別是CD34+細胞的產生,而此結果會增加神經組織中血管生成因子表現,進而促進運動功能的恢復。
Background: Hematopoietic progenitor cells involved in the process of peripheral nerve regeneration occurs mostly through deposits of CD 34+ cells. Treadmill exercise either with various intensity or duration increases axon regeneration and sprouting, but the effect of bone marrow mobilization after treadmill exercise has not been determined.
Material and Methods: Peripheral nerve injury was induced in Sprague-Dawley rats by crushing left sciatic nerve using a vessel clamp. The animals were categorized into two groups either with or without treadmill exercise (20 M/min for 60min per day for 7 days). Cytospin and flow cytometry were used to determine bone marrow progenitor cell density and distribution. Neurobehavior, electrophysiological study, and regeneration marker expression were conducted at one and three weeks after exercise. The accumulation of hematopoietic progenitor cells, immune cells and angiogenesis factors in injured nerves were determined.
Results: Treadmill exercise significantly promoted nerve regeneration. Increased Schwann cell proliferation, escalated neurofilament, and decreased Schwann cell apoptosis were observed 7 days after treadmill exercise. Elevated expression of S-100 and Luxol fast blue as well as decreased numbers of vacuoles were identified in crushed nerves after exercise. Significantly increased numbers of mononuclear cells, particularly CD34+ cells, were induced in bone marrow after treadmill exercise. Deposits of CD34+ cells in crushed nerves paralleled the elevated expressions of Von-Willebrand, Isolectin B4, and VEGF.
Conclusion: Bone marrow hematopoietic progenitor cells especially CD34+ cells can be mobilized by low intensity treadmill exercise and this effect paralleled significant expression of angiogenesis factors and increased motor function.
目 錄
頁次
第一章 前言..............................................1
第二章 文獻回顧...........................................4
2-1 周邊神經系統(nerve system)..............................4
2-1-1 神經元...............................................4
2-1-2 神經幹(nerve trunk)..................................9
2-2 周邊神經損傷.........................................12
2-2-1 周邊神經損傷的原因....................................19
2-2-2 外傷性神經病變
(traumatic neuropathies)............................19
2-2-3 Sedden 分類法.......................................13
2-2-4 Sunderland 分類.....................................14
2-2-5 周邊神經受傷後的改變..................................15
2-3 周邊神經再生過程.....................................16
2-3-1 環境因子............................................16
2-3-2 神經元本身...........................................22
2-4 造血幹/前驅細胞(hematopoietic
stem/progenitor cells;HSCs)........................23
2-4-1 造血幹/前驅細胞與造血系統.............................23
2-4-2 造血前驅細胞的來源....................................24
2-4-3 造血前驅細胞的鑑定....................................25
2-4-4 造血前驅細胞的應用....................................26
2-5-4 造血前驅細胞應用的展望................................27
2-5 人類造血驅細胞移植(hematopoietic
stem/progenitor cell transplantation)..............31
2-6 造血前驅細胞在臨床應用上的瓶頸.........................32
2-7 血管內皮細胞生成因子(vascular endothelial growth
factor,VEGF).......................................32
2-7-1 VEGF生物學效應.......................................32
2-7-2 VEGF對受損神經影響...................................35
第三章 材料與方法.........................................37
3-1 動物模式............................................37
3-2 功能性恢復分析.......................................40
3-3 Cat walk-自動化定量步態分析..........................41
3-4 電生理研究(Electrophysiological study)...............43
3-5 活體內以溴脫氧尿嘧啶核苷標的標記(In vivo
bromodeoxyuridine (BrdU) labeling).................43
3-6 Terminal deoxynucleotidyl transferase-mediate
biotinylated UTP nick end labeling(TUNEL)分析.......44
3-7 西方墨點法(Western blot).............................44
3-8 Cytospin分析........................................45
3-9 Flow cytometric分析.................................46
3-10 免疫組織化學分析.....................................46
3-11 病理學(Histological)實驗.............................47
3-11 統計分析............................................48
第四章 結果..............................................49
4-1 輪帶跑步機運動對於運動功能恢復、電生理與肌肉重量之影響......49
4-2 利用輪帶跑步機運動對於早期與晚期的神經再之影響.............53
4-3 利用輪帶跑步機運動對於骨髓之造血前驅細胞分裂影響...........59
4-4 造血前驅細胞堆積的增加與血管生成因子之表現有顯著的相關性....62
第五章 討論..............................................66
第六章 結論..............................................70
參考文獻..................................................72
圖目錄
頁次
圖一 神經元之類型與構造....................................7
圖二 神經幹包被各種不同之被膜的結構及其相關位置..............11
圖三 基底層的構造.........................................18
圖四 Type IV 膠原蛋白的構造...............................18
圖五 實驗流程圖..........................................39
圖六 坐骨神經功能指數、電生理功能與肌肉重量之評估.............51
圖七 利用CatWalk分析來表現不同時間點之數據..................52
圖八 輪帶跑步機運動7天後,觀察受損神經neurofilament
表現與TUNEL與BrdU分析的結果...........................55
圖九 輪帶跑步機運動後三週,觀察空泡數目、S-100與
Luxol fast blue染色情況.............................57
圖十 輪帶跑步機運動7天後,利用Cytospin分析骨髓抽取物.........60
圖十一 輪帶跑步機運動7天後,觀察受損神經之免疫細胞的沉
澱物與血管生成
因子之表現..........................................63
圖十二 輪帶跑步機運動7天後,以西方墨點法分析方式觀
察神經恢復情況.......................................65
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