神經修復為神經醫學重要課題之一。周邊神經再生需要協助神經相關細胞與神經生長因子作用，例如許旺細胞(Schwann cell)以及神經膠細胞遞送神經滋養因子(glial cell line-derived neurotrophic factor, GDNF)。本篇研究，首先我們結合Cre/loxP系統與桿狀病毒(baculovirus)，建構長效表現神經生長因子GDNF桿狀病毒系統。BacLEGW與BacECre共轉導到大鼠脂肪間葉幹細胞(Adipose-derive stem cell)後，BacLEGW中的神經生長因子表現閘會形成游離微型質體，因此可以延長外源基因在細胞中表現週期。此桿狀病毒長效表現系統能夠有效延長神經生長因子GDNF表現至少20天，並在第二天表現量達到最高峰128 ng/ml，因此能有效吸引神經相關細胞許旺氏細胞遷移，促進神經再生。除此之外，我們也建立了大鼠脂肪幹細胞片製備技術，只需要2天就能完成製備。在動物實驗中，我們植入混成桿狀病毒載體轉導之脂肪幹細胞細胞片至大鼠坐骨神經受損處。實驗結果顯示，此混成桿狀病毒載體轉導之脂肪幹細胞細胞片能夠有效改善以及促進坐骨神經再生，在動物步態行為分析、神經肌肉電位傳導以及預防肌肉萎縮皆有顯著助益。除此之外，藉由免疫染色切片檢驗，我們也能觀察到大量的神經軸突、髓鞘以及血管再生。接著，我們進一步改良桿狀病毒長效表現系統，結合桿狀病毒長效表現系統與CRISPRa建構同時活化多基因之新型桿狀病毒。藉由Baculovirus-CRISPRa系統，我們能夠同時活化脂肪幹細胞基因，促進神經生長因子(BDNF、GDNF與NGF)表現。CRISPRa系統促進脂肪幹細胞表現BDNF、GDNF與NGF能夠吸引許旺氏細胞遷移，並且有效促進背根神經節細胞(Dorsal root ganglion cell)再生，能夠顯著促進神經修復。本篇研究中，我們建構的混成桿狀病毒轉導之脂肪幹細胞細胞片能夠有效改善及促進神經再生，未來將有潛力運用於臨床治療。

Peripheral nerve regeneration requires coordinated functions of supporting cells (e.g. Schwann cells) and neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF), but nerve regeneration is usually far from complete. First, we constructed a Cre/loxP-based hybrid baculovirus (BV) vector which enabled intracellular formation of episomal DNA minicircle for effective transduction of rat adipose-derived stem cells (ASCs). The GDNF expression persisted for >20 days with the peak level (≅128 ng/ml) tremendously exceeding the picogram levels of GDNF secreted by neuroprogenitor cells. The prolonged expression of functional GDNF capable of recruiting Schwann cells and promote nerve regeneration. We further developed a facile method to fabricate and transduce cell sheets composed of undifferentiated ASCs in 2 days for cell sheet fabrication. Implantation of the hybrid BV-engineered, GDNF-expressing ASCs sheets into sciatic nerve transection site in rats significantly improved the nerve repair, as judged from the enhanced functional recovery, nerve reinnervation, electrophysiological functionality, Schwann cells proliferation/infiltration, axon regeneration, myelina- tion and angiogenesis. In second part, we further improve baculovirus long-term gene expression system by combination of baculovirus and CRISPRa for simultaneous activation of multiple genes. By using this baculovirus-CRISPRa system, we successfully activated the neurotrophic factor genes BDNF, GDNF and NGF in ASCs. CRISPRa stimulated the expression of neurotrophin not only induced the Schwann cell migration but also stimulated regeneration of DRGs. These data and features implicate the potentials of ASCs sheets functionalized by the new hybrid BV for peripheral nerve regeneration.

第一章 文獻回顧 4
1-1 神經系統(Nervous system) 4
1-1-1 中樞神經系統(Central Nervous System) 5
1-1-2 周圍神經系統(Peripheral Nervous System) 5
1-2 神經生長因子(Neurotrophic factors) 6
1-3 神經受損與神經修復 6
1-4 脂肪幹細胞應用於細胞療法 7
1-5 幹細胞的特性與在組織工程上之應用 8
1-6 基因治療 9
1-7 桿狀病毒表現系統 12
1-7-1 桿狀病毒之特性 12
1-7-2 桿狀病毒應用於組織工程 14
1-8 Cre重組酶與loxP序列 15
1-9 常間回文重複序列叢集（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR） 17
1-10 研究動機 18
第二章 材料與方法 27
2-1 重組桿狀病毒之建構與製備 27
2-1-1 昆蟲細胞培養 27
2-1-2 桿狀病毒長效表現載體(donor plasmid)之建構 27
2-1-3 重組表現載體之轉置反應(transposition) (Bac-to-Bac system) 28
2-1-4 重組bacmid之分離 28
2-1-5 重組bacmid之轉染反應(transfection) (製備p0病毒) 29
2-1-6 基因重組桿狀病毒放大培養 29
2-1-7 病毒感染效價測定(infectious titer) 30
2-2 桿狀病毒搭載CRISPRa系統質體建構 30
2-3 大鼠脂肪間葉幹細胞之分離與培養 31
2-4 大鼠背根神經節細胞(dorsal root ganglion cells, DRGs) 之分離與培養 32
2-5 基因重組桿狀病毒之轉導 34
2-6 酵素免疫分析(ELISA)生長因子之含量 34
2-7 細胞遷移實驗(Migration assay) 35
2-8脂肪幹細胞片(Stem cell sheet)製備 36
2-9 背根神經節細胞 (dorsal root ganglion cells) 再生實驗 36
2-10 脂肪幹細胞片手術植入大鼠坐骨神經受損處程序 38
2-11 動物行為步態分析 38
2-12 神經肌肉電生理測試 38
2-13 肌肉量測量 39
2-14 神經組織切片染色分析 39
2-14-1 H&E與Toluidine blue染色 39
2-14-2 組織免疫染色 39
2-15 統計學分析 40
第三章 實驗結果 (I) 45
以桿狀病毒改質脂肪幹細胞片長效表現外源性神經生長因子GDNF改善神經再生 45
3-1 建構基因重組桿狀病毒與脂肪間葉幹細胞(ASCs)神經生長因子GDNF表現分析 45
3-2 分析ASCs表現GDNF誘導許旺氏細胞遷移之效率 45
3-3 桿狀病毒轉導脂肪幹細胞片製備 46
3-4 脂肪幹細胞片表現神經生長因子GDNF改善神經受損後運動功能恢復 47
3-5 長效表現GDNF之脂肪幹細胞片改善神經再支配 48
3-6 運動神經再生預防肌肉萎縮 48
3-7 組織切片染色分析神經再生與血管新生 49
第四章 實驗結果 (II) 57
運用CRISPR系統活化脂肪幹細胞內源性生長因子促進神經再生 57
4-1 搭載CRISPRa SAM系統之基因重組桿狀病毒建構 57
4-2 桿狀病毒轉導後脂肪間葉幹細胞(ASCs)中神經生長因子表現量之分析 58
4-3 CRISPRa系統促進ASCs表現神經生長因子誘導許旺氏細胞遷移 59
4-4 神經生長因子促進背根神經節細胞 (dorsal root ganglion cells) 再生 60
第五章 結果與討論 64
第六章 未來工作 68
6-1 CRISPRa系統促進修復坐骨神經損傷 68
6-2 結合長效表現型基因重組桿狀病毒和CRISPR interference (CRISPRi)系統 70
6-2-1 CRISPRi桿狀病毒轉導後ASCs細胞片之mRNA分析 70
6-2-2 轉導後ASCs細胞片之神經生長因子蛋白表現量分析 71
6-3 CRISPRi桿狀病毒系統轉導ASCs細胞片之定性測試 71
6-3-1 趨化能力測試 71
6-3-2 ASCs細胞片促進背根神經節細胞DRGs再生分析 71
6-3-3 轉導後脂肪幹細胞片促血管新生能力 72
6-4 結合CRISPRa與CRISPRi系統，建構可進行CRISPRa/i調控的桿狀病毒系統 72
第七章 參考文獻 76

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