桿狀病毒已被證實可作為基因載體應用於間葉幹細胞之基因改質。然而桿狀病毒在哺乳動物細胞內不會進行複製，且病毒基因體和選殖基因會隨著細胞的增生，逐漸被稀釋和分解，造成轉殖基因表現期較短。因此本實驗室先前發展了一套具備長期穩定表現轉殖基因的系統，以BacFLP帶有特定點重組酶(Capone et al.) FLP基因，對BacCON-CE的Frt 序列間基因，進行催化反應，協助Frt 序列間的基因重組，形成存在於染色體的游離型態質體，並藉由來自EBV的oriP/EBNA1基因使之隨細胞依同複製。此系統可成功延長轉殖基因表現，並加速刺激幹細胞往硬骨分化，說明長效型桿狀病毒具有骨修復的潛能。為利於長效型重組桿狀病毒日後之發展及應用，本研究的目的為評估長效型桿狀病毒的安全性。我們利用BacFLP及BacCON-CE共同轉導人類間葉幹細胞，並未影響細胞存活及細胞生長速率。再者經長效型桿狀病毒轉導之間葉幹細胞由染色及Q-RT-PCR分析後，證明仍然可往脂肪、硬骨及軟骨分化。以PCR及FISH分析episomal及chromosomal DNA的結果，可發現長效型重組桿狀病毒基因及外源基因不會嵌入至轉導後細胞的染色體中。此外利用Karyotype、qRT-PCR及western blot分析轉導後之人類間葉幹細胞，發現並無染色體對數及結構之缺失或改變，且在抑癌及致癌基因上也沒有過度活化的表現。綜合上述，我們可知長效型重組桿狀病毒為一個安全有效的系統。

第一章 緒論 1
第二章 文獻回顧 4
2-1 桿狀病毒/哺乳動物細胞表現系統 4
2-2 動物細胞中以游離態質體轉殖基因表現技術 5
2-2-1 FLP/Frt系統 5
2-2-2 oriP /EBNA1系統 6
2-2-3 結合FLP/Frt與oriP/EBNA1系統 7
2-3 間葉幹細胞 (Mesenchymal stem cell, MSC) 7
2-4 病毒載體之安全性 8
2-5 研究動機 8
第三章 實驗材料與方法 11
3-1 重組桿狀病毒建構 11
3-2放大及濃縮重組桿狀病毒 11
3-2-1 昆蟲細胞的培養與繼代 11
3-2-2 重組桿狀病毒之放大 12
3-2-3 病毒轉導策略 12
3-2-4 重組桿狀病毒之有效價數 12
3-3 細胞來源與分化 13
3-3-1 人類間葉幹細胞(Mesenchymal stem cell， MSC)之分離 13
3-3-2 轉導人類間葉幹細胞(mesenchymal stem cell，MSC)並誘導分化為軟骨細胞(chondrocyte)、骨細胞(osteocyte)及脂肪細胞(adipocyte) 14
3-3-3 MSC之組織染色法分析 14
3-4 及時偵測同步定量聚合酶連鎖反應(quantitative real-time polymerase chain reaction，Q-RT-PCR分析) 15
3-4-1 細胞RNA的分離 15
3-4-2 即時定量聚合酶連鎖反應 16
3-4-3數據處理及運算 16
3-5 細胞毒性測試(MTT assay) 17
3-6 細胞增生試驗 17
第四章 結果 28
4-1 長效型重組桿狀病毒之安全性探討 28
4-2 長效型重組桿狀病毒轉導對人類間葉幹細胞之細胞特性影響 29
4-2-1 長效型重組桿狀病毒對人類間葉幹細胞之細胞毒性探討 29
4-2-2 長效型重組桿狀病毒對人類間葉幹細胞之生長之影響 29
4-3 長效型重組桿狀病毒對人類間葉幹細胞分化之影響 30
4-3-1 長效型重組桿狀病毒對人類間葉幹細胞分化能力之影響 30
4-3-2 長效型重組桿狀病毒轉導人類間葉幹細胞之特定分化基因 31
4-4 檢驗長效型重組桿狀病毒及外源基因嵌入細胞染色體之可能性 32
4-4-1 聚合酶連鎖反應分析 32
4-4-2 螢光原位雜交分析 32
4-5 探討長效型重組桿狀病毒轉導人類間葉幹細胞之變異 33
4-5-1 轉導後之人類間葉幹細胞之染色體核型分析 33
第五章 討論 42
5-1 重組桿狀病毒對間葉幹細胞之特性影響 42
5-2 檢驗重組桿狀病毒及外源基因嵌入細胞染色體之分析 43
5-3 重組桿狀病毒轉導人類間葉幹細胞之變異 43
第六章 結論與未來展望 45
第七章 參考文獻 46
圖目錄
圖2- 1、出芽型和封埋體之桿狀病毒型態。 1
圖3- 1、FLP/Frt基因重組機制。 1
圖3- 2、重組桿狀病毒Bac-CE示意圖。 1
圖4- 1、桿狀病毒對細胞存活及生長速率之影響 1
圖4- 2、人類間葉幹細胞經桿轉狀病毒轉導後往不同分化路徑之觀察 1
圖4- 3、人類間葉幹細胞經轉導後往不同分化路徑之特定基因分析 1
圖4- 4、長效型重組桿狀病毒及外源基因之分布 1
圖4- 5、人類間葉幹細胞經轉導後之染色體核型分析、細胞狀態及螢光表現 1
圖4- 6、人類間葉幹細胞經轉導後之抑癌及致癌基因表現. 1
表目錄
表3- 1、Q-RT-PCR實驗所使用的引子序列 24
表3- 2、PCR實驗所使用的引子序列 25

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