桿狀病毒/昆蟲細胞表現系統(baculovirus/insect cell expression system)為一重組蛋白表現系統，主要以表現需要轉譯後修飾之蛋白為主。近年來，該病毒已被發現可以進入哺乳動物細胞之中，並表現外來基因，有做為基因治療載體之潛力。本研究主要探討桿狀病毒在軟骨組織工程應用上之可行性，並結合新型生化反應器，促使工程軟骨生長。
基於以上目的，首先，我們須探討桿狀病毒轉導(transduction)是否對軟骨細胞造成影響，因此我們將桿狀病毒轉導後之軟骨細胞接種至PLGA支架上，並於兩相旋轉軸反應器(Rotating-shaft bioreactor, RSB)中培養，觀察工程軟骨的生長是否受桿狀病毒轉導之影響。此外，我們也利用帶有骨生長因子(BMP-2)基因之桿狀病毒(Bac-CB)，誘導軟骨細胞表現BMP-2，進而促使軟骨細胞分泌細胞外間質，以生成軟骨組織。最後，我們結合兩相旋轉軸反應器與Bac-CB轉導之軟骨細胞，依不同培養時間，培養出不同品質之工程軟骨，並探討工程軟骨品質對紐西蘭白兔關節損傷之修復情形。
結果顯示，桿狀病毒轉導之軟骨細胞與未轉導之軟骨細胞在培養過程中其存活率均高達90%，且生成之工程軟骨無論在外觀與細胞外間質含量均無顯著差異，並沒有觀察到桿狀病毒對軟骨細胞造成傷害與影響其生成軟骨組織之能力。此外，以Bac-CB來轉導軟骨細胞，確實可誘導軟骨細胞表現BMP-2，且該生長因子亦對軟骨組織生長有明顯助益。最終，我們將Bac-CB轉導後之軟骨細胞配合兩相旋轉軸反應器所得之不同品質工程軟骨，植入紐西蘭白兔之損傷關節，結果也說明了在反應器中培養三周可得到最佳之工程軟骨品質，且對軟骨組織修復有明顯之效果。綜而言之，結合兩相式旋轉軸反應器與桿狀病毒，在紐西蘭白兔隻動物模式下，確實可做為軟骨組織修復之工具。

Baculovirus/insect cell has been used as protein expression system expressing the protein which needs post-translational modifications. In recent years, baculovirus was reported to be capable of transducing mammalian cells and expressing the foreign genes, implying the potential of baculovirus as a gene delivery vector. In this study, we explored the feasibility that baculovirus as a gene delivery vector transduced into chondrocytes cultured with rotating-shaft bioreactor (RSB) could stimulate cartilage formation.
We seeded the baculovirus-transduced chondrocytes into PLGA scaffold and cultured the chondrocytes/scaffold in the RSB for examining the effects of baculovirus transduction on engineered cartilage growth. Also, we constructed a recombinant baculovirus encoding BMP-2 (Bac-CB) to transduce chondrocytes, thus inducing chondrocytes to produce BMP-2 and stimulating themselves to form cartilage. Finally, we combined RSB and Bac-CB-transduced chondrocytes to form engineered cartilage with different qualities (according to cultured time) and implanted the engineered cartilage in the full-thickness defect of New Zealand rabbit’s knee to explore which quality in the engineered cartilage can repair the full-thickness defect well.
The results showed that baculovirus-transduced and mock-transduced chondrocytes both grew with high viability (90%) and form cartilage similar to native cartilage, indicating that baculovirus transduction did not hinder cartilage formation. Also, the Bac-CB transduced chondrocytes expressed high level of BMP-2, stimulating chondrocytes to secrete large amount of extracellular matrix (ECM) and form engineered cartilage. Moreover, the engineered cartilage with different quality produced by RSB showed different repair. The in vitro culture time dictates the construct properties, cell differentiation status, and subsequent in vivo repair, and constructs derived from 3-week culture result in the best repair. Since baculovirus is generally regarded as a safe vector and no adverse effects were observed in this study, and RSB provides a defined dynamic environment for the maturation of cartilaginous constructs, the combination of baculovirus-mediated gene transfer and RSB culture represents a promising, cost-effective method to produce tissue engineered cartilages for in vivo cartilage repair.

目錄
摘要 I
Abstract II
目錄 IV
圖表目錄 VII
第一章 緒論 1
第二章 文獻回顧 4
2-1 關節軟骨之生理與構造 4
2-2 軟骨組織工程 5
2-3 基因治療 7
2-4 桿狀病毒表現系統 10
2-5 實驗動機及目的 12
第三章 材料與方法 18
3-1 建構與放大重組桿狀病毒 18
3-2 桿狀病毒有效價數的測定 19
3-3 軟骨細胞之取得及培養 19
3-4 桿狀病毒轉導策略 20
3-5 支架之製備方法 21
3-6 3D培養之方法 21
3-7 流式細胞儀之分析 22
3-8 細胞存活率分析 23
3-9 反轉錄聚合酶連鎖反應 23
3-10 即時偵測同步定量反轉錄聚合酶連鎖反應 24
3-11 即時偵測同步定量聚合酶連鎖反應分析 24
3-12 機械應力測試 25
3-13支架中細胞數目與細胞外間質分析 25
3-14 動物實驗方法 27
3-15 組織切片染色 28
3-16 酵素免疫分析法 (ELISA) 29
3-17 組織修復的評分方法 30
3-18 統計分析 30
第四章 結果與討論(1)
--桿狀病毒在軟骨組織工程應用上之初步評估--
4-1 桿狀病毒轉導時間之影響 37
4-2 細胞接種密度之影響 38
4-3 桿狀病毒轉導對軟骨組織生成之影響 39
4-4 分化狀態與EGFP衰退之關係 41
4-5 討論 42
第五章 結果與討論(2)
--桿狀病毒（Bac-CB）與反應器（RSB）之協同作用對軟骨組織生成之影響--
5-1最佳轉導病毒劑量與最佳軟骨細胞代數之評估 49
5-2 RSB與Bac-CB的協同作用（synergistic effects）對工程軟骨之影響 50
5-3細胞外間質之分佈情形 51
5-4基因表現與細胞外間質之成分分析 51
5-5討論 52
第六章 結果與討論(3)
--工程軟骨品質對體內組織修復之影響--
6-1 培養時間對工程軟骨性質之影響 61
6-2培養時間對軟骨細胞特異基因之影響 62
6-3軟骨修復（week 4） 62
6-4軟骨修復（week 8） 63
6-5組織評分（Histologic scores） 64
6-6討論 64
第七章 結論與未來展望 74
參考文獻 76

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