大範圍的骨缺陷在目前臨床骨科醫學上是一個難以克服的難題。先前研究常在具有多分化能力的間葉幹細胞中過表達一些骨修復生長因子去進行大範圍骨缺陷的修復。近幾年發展的CRISPR技術受到相當多人的關注，使用CRISPR系統的優點在於我們只需建構小片段的sgRNA便可以同時調控許多不同的基因，然而目前尚未有人應用CRISPR系統在組織工程中，因此本篇研究希望驗證CRISPR系統在組織工程的應用。先前文獻指出，活化經典Wnt訊息傳遞路徑(Wnt pathway)能夠抑制細胞往脂肪細胞分化，間接促進其往骨分化進行，因此本篇研究，我們運用CRISPRa (SAM)系統活化Wnt pathway，希望藉由抑制ASC/BMSC往脂肪方化的趨勢，提升細胞往硬骨分化的能力。
實驗結果證實，一般桿狀病毒搭載CRISPRa系統搭配Cre/loxP長效表現系統能能夠成功在大鼠(Sprague-Dawley rats) ASC和BMSC中提升Wnt10b mRNA 表現量(339倍及510倍)，然而CRISPRa系統對於β-catenin卻沒有顯著的活化效果。另外，活化Wnt10b可以進一步抑制脂肪相關基因FABP4、PPARγ、CEBPα的表現，並提升骨分化相關基因Runx2、ALP及OPN的表現，然而提升量並不顯著，因此後續實驗我們決定使用較易往骨分化的BMSC，並搭配同樣能激活Wnt pathway 的Foxc2和Wnt10b，發現共表現Foxc2和Wnt10b相較於單一表現Wnt10b在BMSC中有更多的鈣沉積，因此在動物實驗中我們共轉導Bac-Foxc2和Bac-W10b在BMSC中，後將轉導的BMSC植入大鼠頭蓋骨缺陷模型，並看到共同表現Foxc2與Wnt10b的BMSC在術後第12周不管是在骨體積或是骨密度都相對於假性轉導組有明顯提升，約能修復大鼠頭蓋骨缺陷≈17.5%的骨面積。
本研究證實CRISPRa系統可成功的在ASC/BMSC中運作，且藉由調控Wnt pathway可成功抑制細胞往脂肪分化並誘導細胞往骨分化，因此未來可嘗試調控更多基因，增加CRISPR系統在骨組織工程臨床上的應用。

Today critical-sized calvarial bone-defect repair remains a challenging task. CRISPR activation (CRISPRa) is an emerging technology that exploits deactivated Cas9 protein and single guide RNA for programmable activation of endogenous gene expression. Here we developed a hybrid baculovirus vector to express the CRISPRa SAM system for simultaneously activation of multiple genes to modulate both canonical and non-canoical Wnt signaling pathway.
We demonstrated that the expression of CRISPRa system in the rat bone marrow-derived mesenchymal stem cells (rBMSC) and rat adipose-derived mesenchymal stem cells (rASC) enabled the activation of Wnt10b and Foxc2 for several hundred fold.
In particular, the activation of Wnt10b inhibited adipogenesis in rASC by suppressing adipogenic transcription factors PPARγ and C/EBPα.
We also verify that CRISPRa-mediated activation of Wnt10b stimulated the expression of osteogenic differentiation markers such as Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and Osteopontin (OPN) in rBMSC and successfully drove rBMSC fate towards the osteoblast . Implantation of the baculovirus-CRISPRa system-engineered rBMSC successfully accelerated and improved calvarial critical-sized defect (6 mm) healing at 4 weeks after implantation These results show that the CRISPRa system can activate endogenous gene expression either in rBMSC or rASC and potentiate the ability of both to differentiate towards osteogenic pathway and repair calvarial bone defects.

摘要...................2
Abstract...................3
第一章 文獻回顧...................6
1-1骨組織工程介紹...................6
1-1-1骨骼的組成...................6
1-1-2骨細胞與骨修復重建機制...................7
1-1-3骨骼系統常見傷害...................9
1-1-4骨組織工程背景與國內外研究情況...................10
1-1-5骨組織工程的發展...................10
1-2幹細胞在組織工程上的應用...................12
1-3經典Wnt信號通路在骨組織工程上的應用...................13
1-4 基因治療...................15
1-5桿狀病毒表現系統...................16
1-6 重組酶系統...................18
1-7 桿狀病毒在骨組織工程的應用...................19
1-8 CRISPR/CRISPRa系統...................20
第二章 材料與方法...................32
2-1重組桿狀病毒之建構與製備...................32
2-1-1昆蟲細胞培養...................32
2-1-2 DNA引子黏合反應(primer annealing)...................32
2-1-3限制酶(DNA restriction enzyme)反應及接合酶(DNA ligase)反應..32
2-1-4建構重組表現載體(donor plasmid)................33
2-1-5 重組表現載體之轉置反應(transposition) (Bac-to-Bac系統)....34
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...................36
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...................37
2-2大鼠脂肪間葉幹細胞之分離與培養...................38
2-3大鼠骨髓間葉幹細胞之分離與培養...................38
2-4桿狀病毒轉導幹細胞之策略...................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鈣沉積定量分析...................42
2-8 動物頭蓋骨缺陷植入所需之細胞轉導與載體製備...................43
2-9 大鼠頭蓋骨手術植入程序...................43
2-10 電腦斷層掃描分析...................43
第三章 結果與討論...................45
3-1 CRISPRa系統之建構...................45
3-2 Wnt10b、β-catenin在脂肪間葉幹細胞(ASCs)中表現量之分析..45
3-3 Wnt10b、β-catenin在骨髓間葉幹細胞(BMSCs)中表現量之分析..47
3-4 Wnt10b對BMSC成骨分化之分析...................48
3-5 搭配Foxc2和Wnt10b對BMSC成骨分化之分析...................49
3-6 以Gelatin支架搭配BMSC修復大鼠頭蓋骨缺陷之分析...................50
3-7電腦斷層掃描分析...................50
3-8討論..51
第四章 參考文獻..61

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