摘要
利用組織工程重建人工組織是目前最有機會解決器官捐贈不足的方法，為了提高人工組織的存活率，血管網絡扮演著十分重要的角色，目前的血管組織工程研究中，大部份使用的三維水凝膠支架皆無法獨立調控物理與化學特性,因此不同微環境對於血管生成的影響尚未被釐清。本研究中成功萃取高純度的兔子膠原蛋白分子，透過膠原蛋白分子自組裝的特性，形成纖維狀兔子膠原蛋白水凝膠(rabbit collagen hydrogel)，此外，我們利用酪胺修飾兔子膠原蛋白分子後，經由辣根過氧化物(horseradish peroxidase (HRP))與過氧化氫(hydrogen peroxide (H2O2))而形成的孔洞狀的酵素交聯膠原蛋白水凝膠(collagen-Ph hydrogel)。透過這兩種不同交聯方法所形成的具有不同微環境的膠原蛋白水凝膠包裹臍帶血來源集落細胞(human blood-derived endothelial colony-forming cells (ECFCs))和人類白脂肪間質幹細胞(human white adipose mesenchymal stem cell(watMsc))， 利用注射方式植入免疫缺乏鼠皮下，七天取出植入物後，分析血管生成的密度、血管尺寸以及血管組織的大小，探討不同水凝膠微環境對血管生成的影響。透過材料特性的熱穩定性、擴散速率、機械性質、膨潤比、纖維或是孔洞的尺寸以及密度，歸納出血管密度、血管尺寸、血管組織的大小之間的關聯性。實驗結果顯示要有較高血管密度的生成，則選擇具有較低的蛋白質濃度,較高的免疫反應和較快的體內的降解速率的水凝膠，同時也能得到較小的人工血管組織。此外，若要有較大的血管尺寸，則水凝膠需要有較低的降解速率以及較高的免疫反應。透過這些探討，我們可以有效地將這些水凝膠的物理化學特性應用在其他的材料系統上，未來可以根據不同組織對於血管密度的需求不同而精準的調控血管組織的生成。

Abstract
Using tissue engineering to reconstruct artificial tissues is the best way to solve the shortage of organ donation. In order to improve the survival rate of organ transplantation, reconstructing blood vessels network plays an important role. In vascular tissue engineering, most three-dimensional hydrogel can not be independently turned physical and chemical properties, so suitable microenvironment of hydrogel for angiogenesis and vasculargenesis has not been clarified.In this study, high purity rabbit collagen molecules has been successful extracted. Through self-assembly of collagen molecules in nature, the formation of fibrous-like rabbit collagen hydrogel is prepared successafully. In addition, we used tyramine to modify collagen molecules to form enzymatically crosslinked collagen hydrogel (collagen-Ph hydrogel) with hole like structure by adding HRP and H2O2. To evaluate the angiosenesis and vasculargenesis of collagen hydrogels, we mixed human blood-derived endothelial colony-forming cells (ECFCs) and human white adipose mesenchymal stem cell (watMSC) with rabbit collagen hydrogels and collagen-Ph hydrogels with different microenvironment and then injected into immune deficiency mice for evaluation of seven days.The lumen density, the size of lumen, and the size of vascularized tissue were analyzied in hydrogel with different microenvironment. The thermal stability, diffusion rate, mechanical properties, the swelling ratio, microstructure of hydrogels was summarized in the relevance of the size of the number of lumen ,the size of lumen, and the size of vascularized tissue.
The results show that higher density of blood vessels we generated, lower protein concentration, the higher immune response in vivo, faster degradation rate of the hydrogel, and smaller artificial vascular tissue we created.In addition, to have a larger size of vessels in tissue construct, the hydrogel with a lower degradation rate and higher immune response are necessary. Through turning physico-chemical properties of collagen hydrogels, we can effectively apply to other hydrogel systems to generate different vascular density precisely for the demand and reguirement of different tissue and organs.

目錄
致謝 I
摘要 II
Abstract III
第一章、緒論 1
1-1血管組織工程 2
1-1-1目前臨床上的需求 2
1-1-2血管組織工程的現況 2
1-2血管的生成以及研究 4
1-2-1血管網絡的生成:血管新生與血管再生 4
1-2-2內皮細胞在血管生成的訊息傳遞 7
1-2-3共同培養在血管生成的影響 11
1-2-4血管組織工程目前所遇到的瓶頸與解決的方法 19
1-3膠原蛋白的結構與特性 20
1-3-1膠原蛋白的結構 20
1-3-2臨床與研究上所使用的膠原蛋白 21
第二章、材料和實驗方法 24
2-1實驗材料及流程 25
2-2兔子皮膠原蛋白(rabbit collagen)的萃取 28
2-3十二烷基硫酸鈉 - 聚丙烯酰胺凝膠電泳（SDS-PAGE） 29
2-4羥脯胺酸(Hydroxyproline)的定量 29
2-5膠原蛋白功能性檢測 30
2-6膠原蛋白酚羥基(collagen-phenolic hydroxyl conjugates(collagen-Ph conjugate))前驅物的合成 30
2-7氫原子核磁共振儀(H1NMR) 31
2-8膠原蛋白的溶血特性 31
2-9膠原蛋白水凝膠熱穩定性測試 31
2-10膠原蛋白水凝膠的擴散特性 32
2-11膠原蛋白水凝膠成膠時間測定 32
2-12膠原蛋白水凝膠機械性質的檢測 32
2-13膠原蛋白水凝膠的膨潤特性 33
2-14膠原蛋白水濃膠的微結構 33
2-15膠原蛋白體內降解及免疫分析 33
2-16膠原蛋白水凝膠在血管生成的功能性檢測 34
2-17相關係數 34
2-18統計分析 35
第三章、實驗結果 36
3-1兔皮膠原蛋白(rabbit collagen)的萃取與特性檢測 37
3-2膠原蛋白酚羥基(collagen-Ph conjugates)前驅物的合成與溶血測試 38
3-3Rabbit collagen hydrogel和collagen-Ph hydrogel在熱穩定性以及擴散和形貌的比較 39
3-4 Rabbit collagen hydrogel以及collagen-Ph hydrogel在成膠時間以及機械性質的差異 40
3-5探討兩種交聯方式的膠原蛋白在特性上的差異 41
3-6水凝膠在動物體內的降解速率以及免疫反應 43
3-7膠原蛋白水凝膠在體內的血管組織的生成 45
3-8相關係數 48
第四章、討論 51
4-1兔子膠原蛋白分子的萃取與膠原蛋白水凝膠rabbit collagen hydrogel 的特性 52
4-2 酵素交聯膠原蛋白水凝膠的特性 53
4-3膠原蛋白水凝膠的體內探討 55
第五章、結論 59
第六章、圖表 61
第七章、參考文獻 73



圖目錄
圖一、血管生成方式示意圖…………………………………………………………………………………11
圖二、內皮細胞血管生成訊息傳遞示意圖…………………………………………………….…..11
圖三、內皮細胞(ECFC)以及間質幹細胞(MSC)在共同培養的交互作用………..18
圖四 膠原蛋白的結構………………………………………………………………21
圖五、rabbit collagen hydrogel以及collagen-Ph hydrogel的合成…………27
圖六、rabbit collagen hydrogel以及collagen-Ph hydrogel在材料特性以及血管生成的探討…………………………………………………………………………28
圖七、膠原蛋白分子純度鑑定……………………………………………………..61
圖八、rabbit collagen-Ph conjugate的合成與溶血特性………………………...62
圖九、rabbit collagen hydrogel與collagen-Ph hydrogel的成膠機制與水凝膠形貌……………………………………………………………………………………..63
圖十、rabbit collagen hydrogel和collagen-Ph hydrogel的成膠時間以及機械性質的檢測……………………………………………………………………………..64
圖十一、rabbit collagen hydrogel的微結構……………………………………...65
圖十二、collagen-Ph hydrogel的微結構………………………………………….66
圖十三、rabbit collagen hydrogel和collagen-Ph hydrogel的膨潤特性……….67
圖十四、rabbit collagen hydrogel和collagen-Ph hydrogel在動物體內的免疫反應……………………………………………………………………………………..68
圖十五、rabbit collagen hydrogel和collagen-Ph hydrogel在動物體內的血管生成能力………………………………………………………………………………..69








表目錄
表一、膠原蛋白的種類………………………………………………….…………21
表二、血管生成能力-免疫反應-rabbit collagen hydrogel的相關性探討…...70
表三、血管生成能力-免疫反應-collagen-Ph hydrogel的相關性探討………….71
表四、血管生成能力-免疫反應-水凝膠的相關性探討……………………………72

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