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系統識別號 U0026-2208201418223900
論文名稱(中文) 利用高頻超音波監測發炎反應在經由彈性蛋白酶引起之肌腱病變
論文名稱(英文) Monitor inflammation Responses in Elastase-induced Tendinopathy by High Frequency Ultrasound
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 102
學期 2
出版年 103
研究生(中文) 吳宜庭
研究生(英文) Yi-Ting Wu
學號 T96011062
學位類別 碩士
語文別 中文
論文頁數 64頁
口試委員 指導教授-吳佳慶
指導教授-周一鳴
口試委員-王世豪
口試委員-郭立杰
中文關鍵字 肌腱  彈性蛋白  肌腱病變  高頻超音波  發炎反應 
英文關鍵字 Tendon  Elastin  Tendinopathy  High frequency ultrasound  Inflammation 
學科別分類
中文摘要 肌腱是緻密的結締組織,它能夠連結肌肉與骨頭,使得肌肉收縮時能夠使帶動骨骼產生動作,它的結構是由大量的膠原蛋白以及少量的彈性蛋白組成。肌腱病變是骨科臨床常見的疾病之一,肌腱病變是一種慢性的疾病,然而關於肌腱病變的成因與相關之機制目前尚不清楚。然而推測過度使用和過度負荷可能是產生肌腱病變主要的因素,當肌腱損傷後可能會干擾肌腱的癒合,使之產生失敗的癒合反應。注射膠原蛋白酶是目前研究肌腱病變最常使用的動物模型,它會導致急性的肌腱損傷,然而肌腱病變通常是慢性的病理過程所導致。臨床上超音波影像系統常被應用在檢測軟組織相關的病理變化,而高頻超音波影像系統有更高的解析度即能夠檢測到組織結構的細部變化與結構的重塑。在這項研究中,我們的目的是透過在SD大鼠的阿基里斯腱(Achilles tendon)處給予彈性蛋白酶(Elastase, ELNase),使其喪失彈性蛋白纖維(Elastin fiber, ELN fiber)後觀察肌腱病變的病理變化。我的假設為利用彈性蛋白酶模擬腱旁組織(Peritendinous tissue)結構中產生的發炎反應,並進而引起彈性蛋白纖維的降解,並導致肌腱病變的產生。同時藉由高頻超音波影像系統,監測發炎反應的動態過程,並且探討腱旁組織的結構變化與可能參與的發炎機制。在人類肌腱病變檢體中利用維爾賀夫(Venhoff’s)染色觀察到彈性蛋白喪失的情形,並透過梅森三色染色(Masson’s trichrome)發現其膠原蛋白成分產生變化。以在大鼠阿基里斯腱旁處注射彈性蛋白酶,確實能夠引起彈性蛋白的喪失,而給予運動後則會進一步的促進它病理的進展。高頻超音影像的初步結果表明皮下組織腫脹在注射膠原蛋白酶 (Collagenase, COLase)後,以及較無法觀察到其腱旁組織的結構位置;彈性蛋白酶注射後於第7,14,28天均可以清楚的觀察到腱旁組織的位置,而這樣的迴聲變化可能與肌腱發炎的過程有關。而透過組織學方式觀察到處理彈性蛋白酶與膠原蛋白酶後其嗜中性球細胞(Neutrophil)和環氧合酶-2(Cyclooxygenase, COX-2)表達的增加,而膠原蛋白酶所引起嗜中性球的浸潤是較多的,因此在體內方式的結果得知,彈性蛋白酶與膠原蛋白酶所引起的嚴重程度與疾病進展是不一樣的。在體外培養肌腱的結果,彈性蛋白酶引起彈性蛋白的降解,使肌腱結構產生輕微的改變,並刺激肌腱細胞(Tenocytes)表現出環氧合酶-2;而膠原蛋白酶的影響則是嚴重的結構破壞與刺激肌腱細胞的環氧合酶-2大量表達;而無論是在體內和體外的結果通過膠原蛋白酶和彈性蛋白酶對肌腱的影響都類似,因此建議,彈性蛋白酶的給予後,其產生的病理變化與過程,是較能夠符合臨床的病理機制。
英文摘要 The tendinopathy is a common clinical disease. Previous studies, tendinopathy was used COLace injected to induced severe tendon extracellular matrix (ECM) damage. But Tendinopathy was a chronic disease. We use the ELNase injection to simulate inflammation at early stage in peritendinous tissue and induce tendinopathy in chronic stage. In this study, the dynamic inflammation processes were monitored by high frequency ultrasound (HFU) and correlated to the changes of peritendinous structure with possible inflammatory signal pathways. Loss of ELN was observed in human tendinopathy tissue with remodeling of COL compositions by Venhoff’s and Masson’s trichrome stain. The paratendon injection of ELNase successfully induced the loss of ELN and exercise (Ex) after inject ELNase can further facilitated the pathological progresses. Increases of neutrophils in early stage by ELNase and COLase injected. The preliminary results of HFU images demonstrated the dynamic processes of subcutaneous swelling at early stage and change of echogenicity in peritendinous tissue at chronic stage. The results may be related to COX-2 expression. The COX2 expressions were observed in both ELNase and COLase treatments, but showed different severity and progress pattern. The ex vivo incubation of ELNase degraded the ELN fiber and induced the changed of tendon structure. The COX2 production in tenocytes from changed of structure. Both the in vivo and ex vivo results suggested the severe damage of tendon structures by using COLase and the usage of ELNase to tendinopathy is more similar to clinical pathogenesis.
論文目次 目錄
中文摘要 I
英文延伸摘要 Ⅲ
目錄 Ⅶ
圖目錄 Ⅸ
第一章 前言 (Introduction) 1
1.1 肌腱病變的形成 (Tendinopathy) 1
1.2 肌腱損傷與發炎反應 (Tendon injury and inflammation) 2
1.3 肌腱之結構 (Structure of tendon) 4
1.4 肌腱中的彈性蛋白 (Elastin fibers in tendon) 6
1.5 肌腱病變之研究 (Study of Tendinopathy) 7
1.6 高頻超音波系統 (High-frequency ultrasound (HFU) system) 8
目的 (Objective) 10
具體目標 (Specific Aims) 10
第二章 方法與材料 (Materials and Methods) 11
2.1 肌腱病變的動物模型 (In vivo Animal model of tendinopathy) 11
2.2 體外肌腱培養模型 (Ex vivo model) 12
2.3 高頻超音波系統 (High-frequency ultrasound (HFU) system) 14
2.4 組織學分析 (Histological analysis) : 16
2.5 蘇木紫與伊紅染色法 (Hematoxylin and Eosin (H&E) stain) 20
2.6 梅森三色染色法 (Masson’s trichrome stain) 22
2.7 維爾赫夫染色法 (Verhoeff stain) 24
2.8 免疫組織化學染色法 (Immunohistochemical (IHC) staining) 26
2.9 免疫螢光染色法 (Immunofluorescence (IF) stain) 28
第三章 實驗結果 (Result) 30
3.1 彈性蛋白喪失在臨床肌腱病變患者與動物模型之病理變化 30
3.2 藉由體外基件培養條件確認細胞外基質之降解與所引起之發炎訊號 34
3.3 彈性蛋白酶引起之病理變化與可能相關之發炎反應和機制 42
3.4 高頻超音波產生迴聲增強現象與發炎反應之表現 48
3.5 彈性蛋白酶與膠原蛋白酶引起體內肌腱結構的改變 52
第四章 討論 (Discussion) 54
第五章 結論 (Conclusion) 60
第六章 參考文獻 (References) 61
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