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系統識別號 U0026-0902201219495900
論文名稱(中文) 利用高頻超音波研究骨折初期的組織發炎程度
論文名稱(英文) Investigating Tissue Inflammation in Early Stage of Bone Fracture by Using High-Frequency Ultrasound
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 100
學期 1
出版年 101
研究生(中文) 陳彥竹
研究生(英文) Yen-Chu Chen
學號 t96994040
學位類別 碩士
語文別 英文
論文頁數 60頁
口試委員 指導教授-吳佳慶
口試委員-黃步敏
口試委員-王士豪
中文關鍵字 骨折癒合  發炎  高頻超音波  超音波影像  迴聲 
英文關鍵字 fracture healing  inflammation  high-frequency ultrasound  ultrasonography  echogenicity 
學科別分類
中文摘要 骨折癒合過程可分為發炎、軟性骨痂生成、硬性骨痂生成以及組織重建四階段;在骨折癒合初期,適度的發炎反應已被證實有益於損傷組織修復。發炎主要是由花生四稀酸 (AA) 藉由還氧合酶 (COX) 或5-脂氧合酶 (5-LOX) 途徑轉換成前列腺素 (PG) 及白三烯 (LT) 引起,而COX2是發炎反應因子之一,調控釋放PG來調控破骨母細胞的骨吸收作用及成骨母細胞的骨新生成作用;因此檢測骨折癒合前期的發炎是非常重要的,不僅是診斷癒合過程,更可發展治療策略,然而,少有非侵入性的技術被發展並針對骨頭損傷來研究急性發炎。而超音波可藉由迴聲即時偵測組織結構變化,且頻率高於15MHz的高頻超音波具有較佳的解析度來評估細微的組織結構。在本研究中,我們的目的是利用高頻超音波有效評估骨折癒合初期的發炎程度並與組織學檢驗相關聯其發炎反應到組織重建。
我們在麻醉的B6母鼠的左脛骨中段利用自由落體撞擊損傷模擬裝置段給予垂直衝撞力(5.15×10-1N)建立動物骨折模型,在骨折後第一、二、四和八週利用微電腦斷層掃描監控骨頭癒合動態並量測骨痂體積、骨頭體積、組織體積和骨密度;骨頭修復過程中,在第二週有最大的骨痂體積,有最大骨頭體積在第四週,而在第八週的骨折骨中有最高的骨密度表現。因此我們利用50MHz高頻超音波更進一步觀察急性損傷後第零天、第二天、第四天、第七天和第十四天骨折處軟組織的發炎反應;高頻超音波研究結果顯示在第二天、第四天和第七天骨折處週圍的迴聲有顯著增加。將細菌性脂多醣(LPS)利用肌肉注射方法注射到年齡吻合健康老鼠小腿肌肉上,藉此確認局部發炎反應對超音波影像迴聲表現的影響;細菌性脂多醣注射後兩小時,小腿肌肉區域的超音波影像也顯示高強度迴聲。骨折老鼠給予口服餵食阿斯匹靈(Asp)、非選擇性COX抑制劑¬¬¬-indomethathin (IND)、選擇性COX-2抑制劑-SC-236、以及5-LOX抑制劑-Zileuton,來研究發炎抑制劑對早期癒合的影響。結果發現給予Zileuton或Asp治療的骨折老鼠,在骨折後第二天、第四天和第七天的高頻超音波檢測迴聲有降低的現象;並結合組織學實驗發現Zileuton與Asp對骨折癒合的影響在較早的成熟軟骨細胞形成,Asp同時也減少了細胞死亡和降低COX-2及LTB4受體(BLT1)的表現量。總結,我們建立老鼠骨折模型來研究早期骨折癒合發炎反應並利用高頻超音波定量量測組織發炎,更進一步利用超音波影像與組織學的改變相關聯,可以有效的研究不同的治療策略來解釋骨折癒合的早期發炎並應用於未來臨床診斷與治療。
英文摘要 The processes of fracture healing are divided into four phases: inflammation, soft callus formation, hard callus formation and tissue remodeling. Moderate inflammatory responses in early stage of bone fracture have been shown to benefit injured tissue repair. Inflammation was induced by converted arachidonic acid to prostaglandins (PG) or leukotrienes (LT) via cyclooxygenase (COX) or 5-lipoxygenase (5-LOX) pathway. COX-2, a factor participated in inflammatory responses, controls the release of prostaglandins to trigger bone reabsorption and new bone formation in osteoclasts and osteoblasts, respectively. Detection of early inflammation in bone fracture is important not only for diagnosing healing progresses but also for developing treatment strategies. However, only few non-invasive techniques are developed and focused on injured bone for studying the acute inflammations. Utrasound could detect tissue structure changes by echogenicity in real time. A High-frequency ultrasound (HFU), with frequency above 15MHz, is considered to provide better resolution for evaluating details of tissue structure. In this study, our objective is to effectively measure the inflammation levels during early fracture healing by HFU and correlate the inflammatory responses to tissue remodeling by histological examinations.
The animal fracture model was created on the left tibia in anesthetized female B6 mice by applying a vertical impact force (5.15×10-1N) using a free-dropping impacted-injury simulator. To monitor the dynamics of bone healing, the micro-computed tomography was used to evaluate the volume of tissue, callus, and bone and the bone mineral density on 1, 2, 4, and 8 weeks after fracture injury. The highest volume of bone formation was detected at 4 weeks and highest callus volume can be identified as early as on 2 weeks. The highest mineral density in fracture bone was observed after 8 weeks of injury. Therefore, we further observed the soft tissue inflammatory responses by 50MHz of HFU on fracture site at day 0, 2, 4, 7, and 14 after acute injury. Our HFU results showed a significant increase of echogenicity around fracture site on day 2, 4, and 7. To confirm the local inflammation effect on echogenicity expression in ultrsonography, the intramuscular injection of lipopolysaccharide (LPS) was performed to the calf muscles in age-matched healthy mice. The ultrasonography also showed hyperechogenity in calf muscle region at 2 hours after LPS injection. Oral treated with Asprin (Asp), non-selective COX inhibitor (indomethathin, IND), COX-2 selective inhibitor (SC-236), and 5-LOX inhibitor (Zileuton) in the fracture mice to study the inflammation inhibitors effect on early repair. The result showed the treatment of Zileuton and Asp reduced the echogenicity of HFU on day 2, 4, and 7. To combine with histological experiment, Zileuton and Asp influence on frature mice with early mature condrocyte formation. Asp also decreased cell death, COX-2 and BLT1 expression on day 7.
Taken together, we have established a fracture model to investigate the histological changes in related to early inflammatory responses and quantitatively measuring the tissue inflammations by using HFU images in mice. The detail correlation between histological changes in remodeling tissue and the HFU images will be further analyzed with different drug treatments. The therapeutic strategies can be effectively studied for elucidating the early inflammations on healing of bone fracture and have future implications in clinical diagnoses and treatments.
論文目次 中文摘要 III
ABSTRACT V
INTRODUCTION 1
Bone fracture repair 1
Inflammatory response on fracture healing 2
Inflammation inhibitor effect 4
High frequency ultrsound 5
SPECIFIC AIMS 7
MATERIALS AND METHODS 8
Animal object 8
Fracture model in vivo 8
Micro computed tomorgrophy (μCT) 9
High frequency ultrasound (HFU) 11
Inflammation inhibitors treated 13
Local inflammation model in vivo 13
Paraffin embed 14
Hematoxylin and Eosin (H&E) 16
TUNEL assay 18
Immunohistochemistry 20
Statistical analysis 22
RESULTS 23
Determination fracture model repaired process 23
Early fracture repair stage expressed hyperechogenic ultrasonography 30
Hyperechogenic ultrasonography of local inflammation tissue 36
Inflammation inhibitors caused different echogenicity level on inflammatory phase of fracture healing 40
DISCUSSION 49
CONCLUSION 55
REFERENCES 57

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