臺灣博碩士論文加值系統

本研究使用醫用級的聚胺基甲酸酯（PU）-Pellethane 2363-80A為材料，嘗試以不同的加工法，包括混鹽浸鑄法、非溶劑水凝法及混鹽浸鑄水凝法，製作成具微孔洞相連結構之小口徑人工血管；其中混鹽浸鑄水凝法可得到良好機械性質之人工小血管，經電子顯微鏡（SEM）發現其具極佳的微孔洞相連結構，且具有混合式孔隙度。
以混鹽浸鑄法製成人工小血管，並以明膠與含RGD的重組蛋白質CBD-RGD加以改質。以三段式重力法將人臍靜脈內皮細胞株植覆於血管內壁，再以靜態或動態環境下培養，培養一週後，發現細胞並無明顯的生長。進行無血清培養液沖刷實驗，計算細胞殘餘率，可評估其抗沖刷能力，並以SEM作為輔證。結果發現，靜態培養一週後，雖然細胞並無增生，但在沖刷後所得細胞殘餘率為98.9﹪，抗沖刷能力極佳；在1 rpm以上的動態培養一週後，內皮細胞數量減少為一半，在沖刷後細胞殘餘率約僅30％，而在1 rpm以下動態培養的情形有稍微改善，未來應繼續降低轉速，以其找出最佳培養環境。
經明膠改質的內徑3mm、長3∼4 cm人工小血管，在植入豬的頸動脈後，可利用都卜勒超音波觀察血管內部血液流動的情形。經過2個月後血管阻塞，取出人工血管接合處附近動脈血管作組織切片，發現有內膜增生的現象。
自豬體內取得的頸靜脈內皮細胞，體外培養後可達第3代，至第4代細胞會變形呈多觸角狀。經VWF鑑定1及2代仍有內皮細胞之特性。

A medical grade polyurethane, Pellethane 2363-80A, was used to fabricate the sponge type small-diameter vascular grafts in this study, using three different processes, including salt- impregnated, salt-impregnated water-coagulation and non-solvent coagulation. The grafts had an open-cell structure. Among them, those fabricated by salt-impregnated water-coagulation method had the best mechanical properties and composite porosity.
The grafts fabricated by salt-impregnated method were modified with gelatin and CBD-RGD (celluose-binding domain RGD-containing protein). Human umbilical vein endothelial cell lines were seeded on the graft lumen by three-step gravitational method. After being seeded, the grafts were cultured in static or dynamic conditions for a week. The grafts were perfused in vitro for 3 hours and the cell retention was measured. In static conditions, the cell retention was 98.8%. In dynamic conditions, the cell retention was greatly reduced when the rotational speed for the culture deck was above 1 rpm. When the speed was below 1 rpm, the cell retention was better.
The grafts modified with gelatin (3-4 cm in length and 3 mm in diameter) were implanted into porcine carotid arteries. The patency was evaluated by monitering the blood-flow using Doppler-ultrasound. After two months, the thombus was found. The grafts and the surrounding tissues were resected for histological studies. There was intimal hyperplasia observed near anastomotic site.
The porcine jugular vein endothelial cells could be harvested and cultured up to the third passage. After that, the cells became elongated and lost the phenotypic morphology. The cells for the passages 1 and 2 contained VWF antigens on their surface.

中文摘要………………………………………………………………... I
英文摘要………………………………………………………………... II
謝誌……………………………………………………………………… III
目錄……………………………………………………………………… IV
表目錄…………………………………………………………………… VIII
圖目錄…………………………………………………………………… IX
第一章 緒論…………………………………………………………… 1
前言…………………………………………………………………. 1
研究目的………………………………………………………….… 1
第二章 文獻回顧……………………………………………………... 3
2-1 血管的構造…………………………………………………… 3
2-2 血管的病變…………………………………………………… 4
2-2-1 動脈硬化…………………………………………………... 4
2-2-2 動脈瘤……………………………………………………... 5
2-3 人工血管……………………………………………………… 5
2-3-1 生物組織型人工血管……………………………………... 5
2-3-2 合成型人工血管…………………………………………... 6
2-3-3 生物混合型人工血管……………………………………... 9
2-3-4 組織工程型人工血管……………………………………... 10
2-4 人工血管的要求……………………………………………… 11
2-4-1 生物相容性………………………………………………... 11
2-4-2 順應性…………………………………………………….. 12
2-4-3 微孔結構的製作…………………………………………... 14
2-5 人工血管植覆內皮細胞……………………………………… 15
2-6 生物反應器…………………………………………………… 17
2-7 都卜勒超音波………………………………………………… 20
2-7-1 都卜勒效應………………………………………………... 20
2-7-2 彩色都卜勒血流影像……………………………………... 21
第三章 實驗部分……………………………………………………. 23
3-1 實驗藥品……………………………………………………… 23
3-1-1 人工小血管製作…………………………………………... 23
3-1-2 明膠交聯在人工小血管上………………………………... 23
3-1-3 人工小血管作蛋白質序列（RGD）改質……………….. 23
3-1-4 人臍靜脈內皮細胞株（EVC-304）培養液…………….... 24
3-1-5 豬頸靜脈內皮細胞培養液………………………………... 24
3-1-6 磷酸鹽累緩衝液…………………………………………... 24
3-1-7 羥乙基乙烷磺酸緩衝溶液…………………………... 25
3-1-8 取豬靜脈內皮細胞……………………………………….. 25
3-1-9 內皮細胞株（EVC-304）培養與植覆人工血管之培養.... 26
3-1-10 內皮細胞之正立式顯微鏡與電子顯微鏡之觀察………... 26
3-1-11 動物實驗…………………………………………………... 26
3-2 實驗器材……………………………………………………… 27
3-2-1 人工小血管製作…………………………………………... 27
3-2-2 靜態拉伸之機械測試……………………………………... 27
3-2-3 動態機械分析……………………………………………... 28
3-2-4 測量孔隙度……………………………………………….. 28
3-2-5 細胞培養液與緩衝液配製………………………………... 28
3-2-6 內皮細胞株培養與植覆人工血管之培養………………... 29
3-2-7 動物實驗…………………………………………………... 30
3-3 實驗方法……………………………………………………… 31
3-3-1 藥品配製…………………………………………………... 31
3-3-1-1 內皮細胞株（cell line）培養液……………………… 31
3-3-1-2 豬靜脈內皮細胞培養液………………………………. 32
3-3-1-3 磷酸鹽類緩衝液（PBS）……………………………... 32
3-3-1-4 羥乙基乙烷磺酸衝溶液（HBS）…………………. 32
3-3-1-5 三（羥基甲基）胺基甲烷緩衝液（Tris-Buffer）……… 32
3-3-1-6 膠原蛋白（Collagenase）溶液配製……………………. 33
3-3-2 人工小血管的製作………………………………………... 33
3-3-2-1 混鹽浸鑄法……………………………………………. 33
3-3-2-2 混鹽浸鑄水凝法………………………………………. 34
3-3-2-3 非溶劑凝結法…………………………………………. 34
3-3-3 靜態拉伸之機械測試……………………………………... 35
3-3-4 動態機械分析……………………………………………... 35
3-3-5 測量孔隙度（阿基米德法）……………………………... 36
3-3-6 明膠交聯在人工小血管上………………………………... 36
3-3-7 含RGD序列蛋白質改質………………………………… 37
3-3-8 內皮細胞株之培養………………………………………... 37
3-3-9 取豬靜脈內皮細胞………………………………………... 37
3-3-10 內皮細胞植覆人工小血管上與培養……………………... 38
3-3-11 體外沖刷測試（in vitro perfusion）……………………... 39
3-3-12 亞甲基藍染色觀察………………………………………... 39
3-3-13 電子顯微鏡（SEM）觀察……………………………….. 39
3-3-14 動物實驗（蘭嶼小耳豬，25∼35 Kg）…………………. 40
第四章 結果與討論………………………………………………… 41
4-1 人工血管的製備……………………………………………… 41
4-1-1 混鹽浸鑄法………………………………………………... 41
4-1-2 混鹽浸鑄水凝法…………………………………………... 42
4-1-3 非溶劑凝結法……………………………………………... 43
4-2 人工血管植覆內皮細胞與培養……………………………… 44
4-2-1 內皮細胞植覆人工小血管………………………………... 44
4-2-2 經植覆細胞人的工血管之培養………………………….. 45
4-2-2-1 靜態培養………………………………………………... 45
4-2-2-2 動態培養………………………………………………... 46
4-3 動物實驗………………………………………………….…... 47
4-4 豬頸靜脈內皮細胞之培養……………………………….…... 49
第五章 結論與未來方向……………………………….…….……. 50
參考文獻……………………………….…….…………………… 52

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