大範圍的頭蓋骨缺損修復在臨床上是骨科與整形外科醫師的重大挑戰。近年來，幹細胞結合基因治療之新穎技術，已可加速骨修復，而脂肪幹細胞(adipose derived stem cell, ASC)比骨髓間葉幹細胞更容易分離與培養，深具臨床應用潛力。我們已證實使用桿狀病毒轉導ASC使其表現人類骨型態蛋白第二型(bone morphogenetic protein 2, BMP-2)，可以修復兔子股骨的大範圍缺損，但是同樣的轉導策略並無法應用於兔子頭蓋骨大範圍缺損之修復。最近發現某些微小片段RNA (miRNA)具有調控幹細胞分化的能力，因此本研究希望利用miRNA的調控，結合BMP-2的表現，使人類脂肪幹細胞(hASC)被誘導往硬骨路徑分化，並應用於修復頭蓋骨缺損。我們建構了帶有促骨分化miRNA (miR-148b)的桿狀病毒，與FLPo/Frt長效表現BMP-2的桿狀病毒共同轉導hASC後，我們發現細胞內骨分化指標基因表現量有著大幅的提升。之後我們以電腦斷層掃描(CT)分析了長效表現BMP-2結合短效或長效表現miR-148b在裸鼠頭蓋骨缺陷修復的效率，在術後第12周可以修復約九成的缺陷面積與體積，且組織切片染色結果顯示新生骨修復近似於天然的平板骨結構。後續我們證實miR-148b可能藉由調控BMP pathway的抑制劑Noggin (NOG)，以提升hASC的骨分化能力。本研究結合了miR-148b與生長因子BMP-2的共同表現，提升了人類脂肪幹細胞的硬骨分化能力，並成功地將hASC應用於頭蓋骨缺陷之修復，本研究成果對於未來在臨床上的應用應會有相當大的助益。

Bony defects in the craniofacial skeleton remain a major and challenging health concern. Recent studies have shown that baculovirus-mediated gene therapy combined with bone marrow-derived mesenchymal stem cell (BMSC) therapy can improve the calvarial bone healing. Aside from BMSC, adipose-derived stem cells (ASC) have proven to be an abundant source of multipotent stem cells. However, ASC are less superior to BMSC in their osteogenesis potential. Therefore, hereby we aimed to combine baculovirus-mediated microRNA (miRNA) expression with ASC therapy, hoping that the miRNA can assist the regulation of osteogenesis and improve bone healing. We constructed multiple baculoviruses harboring miRNAs putatively associated with osteogenesis, and discovered that miR-148b can up-regulate osteogenic marker gene expression in osteogenic medium. Transduction of human ASC (hASC) with miR-148b and BMP-2, which not only extended the BMP2 expression beyond 14 days but augmented the ASC osteogenesis. Furthermore, the gelatin-coated poly(lactic-co-glycolic acid) (PLGA) scaffold seeded with engineered hASC was transplanted into mice critical size calvarial defect. After transplantation, μCT and histological analyses demonstrated that miR-148b remarkably ameliorated the regeneration of critical size calvarial defect, when compared with the group implanted with hASC only expressing BMP2. The hASC modulated with miR-148b accelerated the bone remodeling and regenerated the bone through the intramembranous pathway, filling ≈90% of the area and volume in 12 weeks. Furthermore, target prediction analysis tools and experimental validation by luciferase 3’ UTR reporter assay identified NOG (Noggin, a BMP antagonist) as a direct target of miR-148b. It was seen that over-expression of miR-148b and BMP-2 leads promotion of BMP-2/p-Smad1/5 signaling than that only expression of BMP-2. This strongly suggests that miR-148b promotes osteogenic differentiation by repressing NOG and its downstream signaling. Our study provides evidence that miRNA-based therapy can be a valuable tool to promote bone regeneration.

摘要 2
Abstract 3
目錄 5
第一章 文獻回顧 7
1-1骨組織工程簡介 7
1-1-1骨組織工程的背景 7
1-1-2骨骼系統 8
1-1-3骨細胞與骨骼的重建機制 10
1-1-4骨骼系統之常見傷害 12
1-1-5大範圍頭蓋骨缺損 13
1-1-6骨組織工程的發展 14
1-2幹細胞的特性與在組織工程上之應用 15
1-2-1脂肪幹細胞 16
1-3基因治療 17
1-4桿狀病毒表現系統 17
1-4-1桿狀病毒之特性 18
1-5 FLPo/Frt重組系統 19
1-6 Cre/loxP重組系統 21
1-7 RNAi系統 21
1-7-1 Small interfering RNA (siRNA) 21
1-7-2 microRNA的生成機制 23
1-7-3 miRNA在骨組織工程中的應用 25
1-8分子影像技術 25
1-9研究動機 26
第二章 材料與方法 35
2-1重組桿狀病毒之製備與轉導 35
2-1-1 昆蟲細胞培養 35
2-1-2 轉殖基因表現載體(donor plasmid)之建構 35
2-1-3 重組表現載體之轉置反應(transposition) (Bac-to-Bac系統) 39
2-1-4 重組bacmid之分離 39
2-1-5 重組bacmid之轉染反應(transfection) (製備p0病毒) 40
2-1-6 基因重組桿狀病毒放大培養 40
2-1-7 桿狀病毒效價測定 41
2-1-8 人類脂肪幹細胞之培養 41
2-1-9桿狀病毒轉導幹細胞之策略 42
2-2基因重組桿狀病毒轉導hASCs後體外分化測試 43
2-2-1即時偵測同步定量聚合酶連鎖反應分析(qRT-PCR) 43
2-2-2酵素免疫分析法(ELISA) 46
2-2-3茜紅素(Alizarin Red)染色分析 47
2-2-4鈣沉積定量 48
2-3 miRNA調控基因分析 48
2-3-1 miR-148b調控基因預測 48
2-3-2 luciferase reporter assay 49
2-3-3西方點墨法(Western blot analysis) 50
2-4細胞存活率與生長分析 52
2-4-1 WST-1細胞存活率分析 52
2-4-2 LlVE/DEAD® Viability/Cytotoxicity Assay 52
2-5細胞載體製備與測試 53
2-5-1 PLGA載體製備 53
2-5-2 動物頭蓋骨缺陷植入所需之細胞轉導與載體製備 54
2-5-3 裸鼠頭蓋骨手術植入程序 54
2-6 Non-invasive骨缺損修復評估 55
2-6-1 電腦斷層掃描 55
2-6-2 H&E染色 56
2-6-3 組織免疫染色 56
2-7統計學分析 57
第三章 結果與討論 58
3-1 先前實驗之數據與結果 58
3-2 CT影像分析頭蓋骨缺陷修復面積 60
3-3 頭蓋骨修復情況與組織切片分析 63
3-4 miR-148b target gene研究 64
3-5 以Cre/ loxP長效系統表現miR-148b並評估其毒性 66
3-6 以Cre/ loxP系統表現miR-148b與BMP-2並應用於動物實驗 69
3-7 頭蓋骨修復情況與組織切片分析 (Cre/loxP重組系統) 70
3-8 結果討論 72
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