大範圍的頭蓋骨缺陷在目前臨床骨科醫學上是一個難以克服的難題。目前人類第二骨形態發生蛋白質(bone morphogenetic protein-2, BMP-2)是公認最有效的骨修復生長因子，然而BMP-2在不同幹細胞誘導骨分化效果並不一致。BMP-2對於骨隨間葉幹細胞(bone mesenchymal stem cells, BMSCs)有良好的骨誘導效果，但是在脂肪間葉幹細胞(adipose-derived stem cells, ASCs) 中單一表現BMP-2仍無法有效的誘導脂肪間葉幹細胞進行骨分化。
先前研究發現Noggin是BMP-2的拮抗劑，會影響ASCs中BMP-Smad信號通路的骨誘導效果。當ASCs中的BMP-2蛋白提升時會產生負回饋機制表現Noggin蛋白，抑制BMP-Smad信號通路。CRISPRi為近年發展迅速之技術，利用失去活性的Cas9蛋白(dCas9蛋白)搭配具有專一性的sgRNA，抑制內源基因表現。本篇研究將結合長效表現BMP-2與CRISPRi系統，用於抑制ASCs中Noggin基因表現，並進一步促進ASCs進行硬骨分化。
經實驗驗證，CRISPRi系統在大鼠(Sprague-Dawley rats) ASCs中能夠有效率抑制Noggin表現量，搭配骨生長因子BMP-2能促進骨分化相關基因Runx2、ALP、OCN及OSX表現，並且在誘導骨分化14天後，可以觀察到實驗組有更多的鈣沉積。動物實驗中，將ASCs共轉導Bac-LEBW及Bac-Sa後貼附於細胞支架，植入大鼠大範圍頭蓋骨缺陷模型，於手術後四周及八周進行分析骨面積、骨體積及骨密度，與NC及Ø組皆有明顯提升。本研究證實長效表現BMP-2搭配CRISPRi系統能夠調控BMP-Smad pathway，誘導ASCs進行骨分化。在未來上可以嘗試運用CRISPR的優勢，建構多組sgRNA進行多基因調控，增加骨組織工程臨床上的運用。

Today critical-sized calvarial bone-defect repair remains a challenging task. Bone morphogenetic protein-2 (BMP-2) is the most potent osteoinductive factors, but is insufficient to potently promote adipose-derived mesenchymal stem cells (ASCs) osteogenesis, probably because Noggin is activated by BMP2 and antagonizes BMP2 during the culture of ASCs. CRISPR interference (CRISPRi) is an emerging technology that exploits deactivated Cas9 protein (dCas9) and single guide RNA for suppression of endogenous gene expression Here we hypothesized that CRISPRi can be harnessed to knock down Noggin expression so as to restore the BMP2 function and promote osteogenic differentiation of ASC.
We demonstrate that the espression of CRISPRi system in ASCs enable suppression of Noggin. We also versify that CRISPRi suppress Noggin expression stimulated the expression of osteogenic differentiation markers such as Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), Osteocalcin (OCN), Osterix(OSX) and Osteopontin(OPN) in ASCs and successfully drove ASCs fate towards the osteoblast. Implantation of the CRISPRi system-engineered ASCs successfully accelerated and improved calvarial critical-sized defect (6 mm) healing at 8 weeks after implantation.
These data altogether confirmed the potential of CRISPRi for suppression of Noggin and promotion of ASC differentiation and repair calvarial ctitical-size defect.

目錄
摘要 2
致謝 4
第一章 文獻回顧 7
1-1骨組織工程 7
1-1-1骨骼的簡介 7
1-1-2骨組織修復重建機制 8
1-1-3骨骼組織常見傷害 11
1-1-4骨組織工程背景 11
1-1-5骨組織工程的發展 12
1-2幹細胞在組織工程上的應用 14
1-3 BMP-Smad信號通路在骨組織工程上的應用 15
1-4基因治療 16
1-5桿狀病毒表現系統 17
1-6 重組酶系統 19
1-7 桿狀病毒在骨組織工程的應用 20
1-8 CRISPR/CRISPRi系統 21
1-9 Orthogonal CRISPR platform 23
1-10 研究動機 23
第二章 材料與方法 33
2-1重組桿狀病毒之建構與製備 33
2-1-1昆蟲細胞培養 33
2-1-2限制酶(DNA restriction enzyme)反應及接合酶(DNA ligase)反應 33
2-1-3建構重組表現載體(donor plasmid) 33
2-1-4 DNA引子黏合反應(primer annealing) 34
2-1-5 重組表現載體之轉置反應(transposition) (Bac-to-Bac系統) 35
2-1-7 重組bacmid之轉染反應(transfection) (製備P0病毒) 36
2-1-8 基因重組桿狀病毒放大培養 37
2-1-9超高速離心濃縮桿狀病毒 37
2-1-10桿狀病毒效價測定 38
2-2大鼠脂肪間葉幹細胞之分離與培養 38
2-3桿狀病毒轉導幹細胞之策略 39
2-4 酵素免疫分析法(ELISA) 39
2-5即時偵測同步定量聚合酶連鎖反應分析(qRT-PCR) 40
2-6茜紅素(Alizarin Red)染色分析 42
2-7 動物頭蓋骨缺陷植入所需之細胞轉導與載體製備 43
2-8大鼠頭蓋骨手術植入程序 43
2-9 電腦斷層掃描分析 44
2-10 組織免疫染色 44
第三章 結果 46
3-1 . Orthologues CRISPRi系統建構 46
3-2 CRISPRi系統抑制Noggin效率分析 47
3-3抑制Noggin對ASCs骨分化相關基因表現 48
3-4 抑制Noggin對ASCs成骨分化之分析 49
3-5 以Gelatin支架搭配ASCs修復大鼠大範圍頭蓋骨缺陷之分析 49
3-6 組織切片染色分析頭蓋骨組織新生 50
第四章 討論 57
第五章 參考文獻 60

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