臺灣博碩士論文加值系統

在組織工程中，發展血管組織進而修復器官及組織是現今醫學工程及再生醫學重要的課題，許多研究指出，一個組織工程良好的修復，在體內不僅僅需要依靠細胞、支架和訊息傳遞，也需要支架良好的血管化。同時證明，間葉幹細胞（Mesenchymal stem cells，MSCs）能有效幫助內皮細胞進行血管新生。本研究目的在共培養臍帶血來源之內皮前驅細胞（Endothelial progenitor cell，EPCs）以及間葉幹細胞，探討兩種細胞之中互利共生關係，並找出最佳共培養比例，再將細胞培養在使用靜電紡絲製備之生物可降解高分子支架聚3-羥基丁酯-co-3-羥基戊酯（Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV），探討細胞在生物高分子之生長情形與在血管組織工程之應用潛力。
首先EPCs以及MSCs從臍帶血經過利用Ficoll密度梯度離心法分離臍帶血之EPCs與MSCs後經過形態學、表面抗原及功能性分析鑑定。探討以不同比例之EPCs 與MSCs 共培養於2D平面之生長情形並評估共培養細胞形成血管能力以及遷移能力，再利用QPCR儀器以及ELISA儀器評估共培養細胞之基因表現量和蛋白質分泌量。接下來再將細胞共培養在3D之PHBV中，利用SEM以及免疫染色分析探討細胞形態學與細胞之間的比例，使用WST-1 assay評估細胞存活率與生長能力，最後再將共培養細胞結合生物高子支架進行動物實驗，探討支架及細胞在體內情形。
2D平面結果顯示，細胞在經過共培養後，MSCs能增強細胞遷移能力以及傷口修復，並且能釋放特殊的細胞因子促使細胞爬移，QPCR結果顯示，MSCs能增強EPCs的細胞分化增生能力，也能促進EPCs產生血管生成因。3D結果顯示，共培養的細胞能夠很好的生長在PHBV生物薄膜上，並且細胞能順向生長。結合以上結果，共培養EPCs以及MSCs能促進血管新生並能應用於血管組織工程發展。

關鍵字: 聚羥基烷酯、內皮前驅細胞、間葉幹細胞、血管組織工程。

Currently, repair and regeneration of tissue by engineered vascular tissue is an important research topic for biomedical engineering and regenerative medicine. Many studies indicated that growth of tissue not only depended on cell, scaffold and signal but also depended on vascular to supply nutrients and oxygen. In addition, many studies also demonstrated that mesenchymal stem cells (MSCs) can support and enhance endothelial progenitor cells (EPCs) to form angiogenesis. The aim of this study was to co-culture cord blood derived EPCs and MSCs on the 2-D surface (tissue plate) and 3-D scaffold to explore the application potential for vascular tissue engineering.
In this study, EPCs and MSCs were harvested from umbilical cord blood by density gradient centrifugation and the cells were recognized by morphology, surface antigen and functional analysis. Firstly, the symbiotic relationship between EPCs and MSCs were investigated on the 2-D surface and find the best co-culture ratio. Then, the MSCs and EPCs were co-cultured in the 3-D scaffold, electrospinning poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to investigate the behavior of cells, including cell growth and viability (by WST-1 assay), blood vessel forming capability, cell migration capability, gene expression and protein secretion with various ratios of co-cultured EPCs and MSCs. In addition, cell morphology also was checked using SEM and immunostaining analysis. Finally, the best ratio of MSCs and EPCs culture in the PHBV scaffold will implant into animal model to check in vivo function.
The 2D results showed that the growth rate of EPCs co-cultured with MSCs can grow faster than EPCs only and wound repairing rate can also be speeded up. Tube formation and QPCR results demonstrated that the two of best cell ratios of EPCs and MSCs for angiogenesis is 60 to 40 and 40 to 60. The 3D result show that the cell can grow in PHBV bio-membrane well. The SEM and immunostaining analysis demonstrated that cells can grow on PHBV orientation. Based on the above results, co-culture of EPCs and MSCs can effectively enhance neovascularization and would be promising cell sources for vascular tissue engineering.

Keywords: Poly(hydroxylalkanoates), Endothelial progenitor cells, Mesenchymal stem cells, Vascular tissue engineering.

目錄
摘要 I
Abstract III
誌謝 V
第一章、緒論 1
1.1 研究動機與目的 1
1.2 研究架構 3
第二章、文獻回顧 5
2.1 幹細胞（Stem cell）簡介 5
2.1.1 幹細胞定義 5
2.1.2 幹細胞分類 6
2.1.3幹細胞來源 7
2.2 內皮前驅細胞（Endothelial progenitor cell，EPCs）簡介 10
2.2.1 內皮前驅細胞 10
2.2.2 內皮前驅細胞之鑑定 13
2.2.3 內皮前驅細胞之功能機制 16
2.3 間葉幹細胞（Mesenchymal stem cells，MSCs）簡介 18
2.3.1 間葉幹細胞 18
2.3.2間葉幹細胞之鑑定 19
2.4細胞外間質 (Extracellular matrix，ECM) 20
2.4.1纖維連接蛋白(Fibronectin) 21
2.5血管組織工程 21
2.5.1血管組織 23
2.6生物可降解材料 25
2.6.1聚羥基烷酯（poly(hydroxylalkanoates)，PHAs）簡介 25
2.6.2靜電紡絲製程（Electrospinning process） 27
2.7表面改質 28
2.7.1電漿處理 29
第三章、實驗材料與方法 30
3.1 實驗儀器 30
3.2 實驗藥品及耗材 31
3.2.1基礎培養基 31
3.2.2細胞激素 31
3.2.3流式細胞儀相關試劑 32
3.2.4 免疫染色分析相關抗體及藥品 33
3.2.5其他實驗藥劑 34
3.2.6實驗耗材 34
3.3培養動物細胞之基本技術 35
3.4 純化人類臍帶血單核細胞（Mononuclear cell，MNC） 36
3.5細胞來源 36
3.5.1建立內皮前驅細胞 36
3.5.2建立間葉幹細胞 39
3.6血球計數盤計數 (Neubauer hemocytometer) 40
3.7內皮前驅細胞與間葉幹細胞之共培養比例最適化 40
3.8細胞遷移能力 41
3.8.1細胞移行能力 (Migration) : 41
3.8.2侵入能力(Invasion) 41
3.9形成血管網路結構的能力 42
3.10細胞表面抗原分析(Cell surface antigen analysis) 43
3.11即時定量聚合酶連鎖反應（Quantitative real-time polymerase chain reaction，QPCR） 44
3.11.1 RNA萃取 45
3.11.2反轉錄聚合酶連鎖反應RT-PCR 46
3.11.3 即時定量聚合酶連鎖反應QPCR 46
3.12薄膜製備 49
3.12.1溶液配置 49
3.12.2電紡薄膜製備 49
3.12.3薄膜表面改質 49
3.12.4培養細胞於生物膜材上 50
3.13細胞存活率分析 WST-1 Assay 51
3.14細胞在生物薄膜上的型態 52
3.14.1免疫螢光染色 (Immunofluorescence staining) 52
3.14.2電子顯微鏡（Scanning Electron Microscope，SEM）細胞型態分析 53
第四章、實驗結果與討論 55
4.1內皮前驅細胞與間葉幹細胞之建立與分析 55
4.1.1內皮前驅細胞之鑑定 55
4.1.2間葉幹細胞之鑑定 57
4.1.3長時間培養-生長曲線 58
4.2共培養內皮前驅細胞以及間葉幹細胞 60
4.3共培養內皮前驅細胞以及間葉幹細胞之功能性分析 62
4.3.1 細胞遷移能力( Migration & Invasion ) 62
4.3.2血管網路形成能力 65
4.3.3特定基因表現分析 65
4.4共培養內皮前驅細胞以及間葉幹細胞於聚羥基烷酯電紡絲支架上 68
4.4.1免疫螢光染色分析 68
4.4.2 SEM 細胞表面型態分析 75
4.4.3細胞存活率分析 WST-1 Assay 78
第五章、結論與未來工作 80
5.1截至目前結論 80
5.2未來工作 81

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