臺灣博碩士論文加值系統

摘要　
　　
　　化學處理與酵素處理為目前常見的去細胞方式，可有效破壞宿主細胞的細胞膜結構，使宿主細胞凋亡，但缺點是會對植入物本身的細胞外間質造成影響，甚至破壞細胞外間質的組成；若細胞外間質成分間的微結構產生變化，則會改變植入物本身的力學性質。另外化學處理與酵素處理的殘留物質，也會促使免疫反應的發生，使移植失敗的機率提高，其生物毒性也會對個體造成危害。本研究背景為避免現階段去細胞方法之缺點，欲利用超音波震盪設備，由頻率範圍介於28至80KHz之超音波能量，對豬隻心臟血管進行去細胞處理，採用單軸拉伸測試，觀察超音波處理後試樣的力學性質變化，以及從膠原蛋白與彈性蛋白的生化定量分析，確認超音波去細胞處理後試樣的細胞外間質成分比例，藉由H&;E與DAPI組織染色切片之結果，評估不同頻率大小的超音波能量與超音波傳導膠的使用，對試樣的去細胞效應。現階段已確認在長時間、高功率輸出以及搭配脈衝模式之條件下，對於豬隻主動脈會有較佳的去細胞效果；在右冠狀動脈的測試實驗，搭配實驗室標準去細胞流程，並選擇適當之反應介質，可使DNA殘餘率降至47%，且能維持細胞外基質的完整性，並保持其力學性質。

Abstract　　
　　
　　Chemical and the enzymatic treatment are popular in decellularization because they could disrupt the membrane of host cell efficiently, and lead to cell lysis. But it may make the effect on extracellular matrix. For example, it will denature the collagen, the main ingredient of ECM. Besides, it may also remove the glycosaminoglycan (GAGs), and break the composition of ECM. If we change the microstructure between the components in ECM, it will also influence the mechanical properties of implant. The residual ingredient of the chemical and enzymatic treatment in implant may lead to immune response, and raise the rate of failure for transplant. The toxicity of implant with using these treatments may harm the health of body. Our research‘ s background is to avoid the shortcoming of the current decellularization, and we intend to use the equipment of sonication with the range of frequency about 28KHz to 80KHz,for decellularization of porcine vessel in the cardiac cycle. We use the uniaxial tensile strength test to investigate the changing of mechanical properties of porcine vessel before and after the decellularization. The biochemical quantitative analysis in collagen and elastin to observe the composition of ECM, and we use the H&;E stain to ensure the efficiency of the ultrasonic decellularization with ultrasound gel.Now we found that in the parameter of long duration, the higher output power and pulse mode with the aota of US decellularization, it can clean the smooth muscle cell in the wall. In the test of right right coronary artery, we combined the standard protocol of decellularization, and choose the medium, we got the DNA residual ratio about 47%.The decell sample also have the mechanical properties before the US decellularization.

目錄

謝誌 I
摘要 II
Abstract III
目錄 IV
圖目錄 VII
表目錄 X
第一章　前言 1
1.1研究背景 1
1.2研究動機 1
第二章　文獻回顧 3
2.1動脈血管去細胞之臨床需求性 3
2.2血管管壁分層構造 4
2.3細胞外基質成分 5
2.4動脈之微結構 8
2.4.1動脈血管三大分層之主要成分與結構 8
2.4.2動脈內彈性蛋白層與彈性蛋白板之結構 9
2.4.3動脈內膠原蛋白束的分布與排列 10
2.5動脈力學性質簡介 12
2.6各種去細胞方式簡介 13
2.7利用超音波能量進行去細胞流程 15
2.7.1超音波概述 15
2.7.2低頻超音波之物理效應 17
2.7.3超音波產生之空蝕效應 18
2.7.4檢測超音波空蝕效應 19
2.7.5利用超音波進行去細胞研究之整理 20
第三章　研究器材與實驗方法 37
3.1實驗藥品 37
3.2儀器設備 38
3.3研究方法 39
3.3.1實驗設計－超音波去細胞之前測試 39
3.3.2實驗設計－血管組織進行超音波去細胞 41
3.4藥品配置 45
3.5組織獲取 46
3.6力學測試 46
3.6.1軸向拉伸 47
3.6.2環向拉伸 47
3.6.3應力－應變圖分析 47
3.7 生化組成分析 48
3.7.1膠原蛋白 (Collagen) 定量分析 48
3.7.2彈性蛋白 (Elastin)定量分析 49
3.7.3醣胺蛋白(Glycosaminoglycan, GAGs)定量分析 49
3.8 DNA純化及定量分析 50
3.9組織型態及細胞核之觀察 51
3.9.1 Hematoxylin and eosin stain (H&;E stain) 51
3.9.2 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI) Stain 51
3.10 使用ImageJ進行細胞計數分析 51
第四章　結果與討論 53
4.1使用鋁箔進行超音波去細胞前測試 53
4.1.1超音波水槽內能量分布測試結果 53
4.1.2小面積鋁箔模擬血管組織使用超音波進行去細胞測試結果 56
4.1.3空蝕效應之鋁箔測試討論 56
4.2使用主動脈進行超音波去細胞 58
4.2.1超音波頻率對去細胞效果之影響 58
4.2.2反應時間長短 58
4.2.3超音波輸出功率大小 59
4.2.4連續／脈衝模式 60
4.2.5反應位置 60
4.2.6使用培養皿固定 61
4.2.7反應介質種類 61
4.3右冠狀動脈進行超音波去細胞 62
4.3.1反應時間 62
4.3.2浸泡異丙醇（Isopropyl alcohol）與trypsin處理之差異 62
4.3.3不同種類超音波介質 63
4.4實驗討論 66
第五章　結論 70
Reference 136

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