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研究生： 白乙辰
Bai, Yi-Chen
論文名稱： 結合非病毒基因工程改造幹細胞與三維生物列印支架用以促進傷口癒合
Integration of Nonviral Genetically-Engineered Stem Cells and 3D Bioprinted Scaffolds for Promoting Wound Healing
指導教授： 張建文
Chang, Chien-Wen
口試委員: 張晃猷
Chang, Hwan-You
江啟勳
Chiang, Chi-Shiun
劉倬昊
Liu, Zhuo-Hao
學位類別： 碩士
Master
系所名稱： 原子科學院 - 生醫工程與環境科學系
Department of Biomedical Engineering and Environmental Sciences
論文出版年： 2018
畢業學年度： 106
語文別： 中文
論文頁數： 101
中文關鍵詞： 三維生物列印 、傷口癒合 、非病毒基因傳遞 、血管內皮生長因子 、人類間質幹細胞
外文關鍵詞： 3D bioprinting, Wound healing, Non-viral gene delivery, VEGF, Stem cell
相關次數： 點閱：2  下載：0 
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    • 　　近年來，人類間質幹細胞(human mesenchymal stem cells, hMSCs)在傷口癒合與皮膚組織再生之應用日益受到關注。hMSCs可以分泌各種生長因子以利傷口癒合，例如：有助於刺激傷口床血管新生之血管內皮生長因子(VEGF)。然而，僅由hMSCs所分泌之VEGF仍不足以增進傷口修復。為此，本研究沿用實驗室所開發之高效率磁性複合奈米基因載體(PNT)，並以磁轉染程序針對hMSCs進行基因工程(VEGFhMSCs)，突破傳統非病毒載體基因傳遞效率不佳之疑慮，作為一安全且有效率之基因遞送平台以加速傷口修復。此外，為解決臨床上移植MSCs之細胞存活度不佳等問題，我們欲結合細胞治療與敷料作為促進傷口修復之策略，利用三維生物列印技術改善二維細胞敷料其細胞分散性低、無法有效獲得養分與代謝而影響細胞活性及遷徙能力之缺點，提供一良好平台遞送細胞。
    　　本研究利用具可列印性且具光交聯性質之材料：GelMA (gelatin methacrylate)與GCMA (glycol chitosan methacrylate)，以及三維生物列印(Three dimensional bioprinting, 3D bioprinting)之技術包覆非病毒基因工程改造後之VEGFhMSCs，製備三維結構之幹細胞水凝膠敷料。將Gelatin與Glycol chitosan經由甲基丙烯酸化後得到GelMA與GCMA，可透過 1H-NMR測定其接枝率分別為75.5％和42.5％。於405nm之光照射3-5分鐘，以製備GelMA/GCMA水凝膠。透過掃描式電子顯微鏡觀察水凝膠的孔洞大小，發現水凝膠的吸水倍率與GelMA/GCMA水凝膠的濃度成反比。與此同時，利用本實驗室所開發的奈米基因載體製備VEGFhMSCs。與市售Lipofectamine 2000相比，由PNT磁轉染的VEGFhMSC中擁有較高的VEGF表達量。進一步探討幹細胞與GelMA/GCMA水凝膠的材料成分比例之間的關係。透過調整水凝膠高分子材料組成分之濃度，可調控水凝膠的結構與孔洞大小，進而提高包覆於水凝膠內之VEGFhMSCs transgene的表達，改善因剪切作用而降低轉染效率之限制，使轉染後細胞具備VEGF表達量最佳化。由水凝膠包覆的VEGFhMSC所分泌的VEGF protein可以在體外有效地提高HUVEC的細胞增生。最後，由體內全皮層受傷模式實驗的結果證實，以此敷料治療之組別，因VEGFhMSCs所分泌之VEGF protein能夠縮短傷口中細胞分裂的週期、刺激真皮層細胞增生及自我更新，達到促進傷口癒合之療效。

    　　Applying human mesenchymal stem cells (hMSCs) on wound healing and skin regeneration have received increasing attention in recent years. hMSCs can secrete various growth factors such as vascular endothelial growth factor (VEGF) which could contribute to neovascular regeneration. However, low viability of the transplanted hMSCs in the inflammatory microenvironment has significantly limited their beneficial effects on wound healing. With the advance of recent 3D printing techniques, it is possible to design biomimicking scaffolds capable of improving survival, proliferation and migration of the transplanted cells. The aim of this study is to develop photocrosslinkable gelatin methacrylate (GelMA)/glycol chitosan methacrylate (GCMA) 3D-bioprinted scaffolds incorporated with VEGF-expressing hMSCs (VEGFhMSCs) as a novel stem cell-based dressing for wound healing. GelMA and GCMA were synthesized and characterized by 1H-NMR. Degree of methacrylation of the GelMA and GCMA were 75.5 % and 42.5 % respectively. GelMA/GCMA hydrogels were fabricated by photo-crosslinking at 405 nm for 3-5 minutes. Porosity of these hydrogels were characterized by scanning electron microscope (SEM). In accordance to SEM results, the swelling ratios of hydrogels were inversely proportional to the content of GelMA/GCMA hydrogels. In parallel, we have developed a magnetic polymer/iron oxide nanocomplex (PNT) as an efficient and non-toxic gene delivery tool for human stem cells. VEGFhMSCs were successfully constructed by PNT-mediated VEGF gene delivery. Much higher VEGF expression level was detected from the hMSCs transfected by PNT compared to Lipofectamine 2000 or polyethylenimine. Inter-talks between stem cells and material properties/composition of the hydrogels were further studied. Higher degree of cell spreading and proliferation were observed from hydrogels containing more GelMA. VEGF secreted from the 3D-encapsulated VEGFhMSCs could effectively enhance the proliferation of human umbilical vein endothelial cells (HUVECs) in vitro. Most importantly, the preliminary in vivo results of subcutaneous transplantation model suggested that the secreted VEGF protein from encapsulated VEGFhMSCs thickens the epidermis, promotes the cell proliferation of dermis that enhances the overall wound healing process.

    摘要 3 Abstract 5 目錄 7 圖目錄 11 表目錄 14 第一章、緒論 15 1.1 前言 15 1.2 研究動機與目標 16 第二章、文獻回顧 19 2.1 全皮層皮膚創傷 19 2.1.1 全皮層創傷修復 19 2.1.2 常見皮膚創傷之治療 23 2.1.3 人類間質幹細胞於皮膚創傷修復策略 26 2.2 基因載體 28 2.2.1 基因傳遞概述 28 2.2.2 病毒基因載體 29 2.2.3 非病毒基因載體 30 2.2.4 基因治療與血管新生 34 2.3 三維生物列印 38 2.3.1 二維與三維生物支架之比較 38 2.3.2 三維生物列印技術之種類 39 2.3.3 應用於擠壓型三維生物列印之生物墨水 43 第三章、實驗材料與方法 47 3.1 實驗材料 47 3.2生物墨水之製備與分析方法 48 3.2.1 Methacrylated Glycol Chitosan (GCMA)的製備 48 3.2.2 Methacrylated gelatin (GelMA)的製備 49 3.2.3 核磁共振光譜分析 49 3.2.4水凝膠之製備 49 3.2.5水凝膠橫切面之結構分析 50 3.2.6水凝膠之Swelling ratio測定與分析 50 3.3生物墨水之體外細胞實驗檢測 50 3.3.1細胞培養與繼代 50 3.3.2細胞於水凝膠內之活性檢測 51 3.4載體製備與純化 52 3.4.1 SPIONs合成與純化 52 3.4.2 γPGA-SPIONs製備與純化 52 3.4.3 PAE與PNT polyplexes載體製備 53 3.5 VEGF製備及鑑定 53 3.5.1 VEGF plasmid DNA的純化 53 3.5.2膠體電泳分析VEGF plasmid DNA 53 3.6 PNT polyplexes於hMSCs之磁轉染與分析 54 3.6.1基因傳遞與蛋白質定量 54 3.6.2磁轉染對細胞毒性之測試 54 3.7結合磁轉染細胞與生物墨水之分析 55 3.7.1結合磁轉染細胞與生物墨水之細胞活性檢測 55 3.7.2體外內皮細胞增生分析 55 3.7.3即時定量聚合酶連鎖反應 55 3.8三維生物敷料於體內實驗測試 56 3.8.1三維生物列印系統 56 3.8.2全皮層傷口動物模式之建立與治療 56 3.8.3犧牲與取樣 57 3.8.4病理切片與染色 57 3.9數據分析 58 第四章、實驗結果與討論 59 第一部分、材料的合成與鑑定 59 4.1 GelMA與GCMA材料的製備與鑑定 59 4.2 GelMA與GCMA水凝膠的製備與鑑定 61 第二部分、探討hMSCs與生物墨水成分之最佳化條件 63 4.3 GelMA的成分比例與hMSCs的交互關係 63 4.4探討水凝膠所使用的培養基成分與包覆於水凝膠內部hMSCs之關係 65 4.5探討於不同成分比例之水凝膠中hMSCs的細胞活性 67 第三部分、探討基因改質hMSCs與墨水材料之交互作用 68 4.6磁轉染於hMSCs之探討 68 4.6.1 VEGF plasmid DNA的純化與鑑定 68 4.6.2磁轉染於hMSCs之參數最佳化 69 4.7探討墨水材料與轉染後hMSCs之關係 72 4.7.1探討2D與3D細胞培養對VEGF transgene表達之影響 72 4.7.2探討VEGFhMSCs包覆於不同比例的GelMA/GCMA水凝膠中的細胞活性 73 4.7.3探討於不同材料的水凝膠中VEGFhMSCs之VEGF transgene的表達 75 4.7.4探討VEGFhMSCs於2D及3D培養下的VEGF基因表現量 76 4.7.5以HUVEC的增生率測定VEGFhMSCs所分泌的VEGF protein功能性 77 第四部分、結合VEGFhMSCs與水凝膠敷料應用於傷口癒合 78 4.8結合VEGFhMSCs與水膠敷料於傷口癒合應用之結果 78 4.8.1外部傷口的縮合 78 4.8.2傷口內部組織的H&E及Masson trichrome染色 80 4.8.3傷口內部組織的Human nuclei與CD31抗體染色 83 第五章、結論 86 參考文獻 87

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