摘要
大範圍骨缺陷通常不易痊癒且治療方式複雜，牽涉到許多困難的外科手術。近來，結合病毒基因載體與間葉幹細胞療法發展出之新穎技術，已顯示可協助並加速骨修復。其中脂肪幹細胞(ADSCs)由於容易分離培養，近來亦受到廣泛研究應用。本研究目的探討藉由FLP/Frt基因延長表現系統之新型重組桿狀病毒轉導ADSCs後是否可以加速修復大範圍骨缺陷。首先利用帶有FLP/Frt系統的基因重組桿狀病毒Bac-FCBW(表現第二型骨型態蛋白(BMP2))及Bac-FCVW(表現血管內皮細胞生長因子(VEGF))轉導ADSCs，觀察表現BMP2或VEGF能力。結果顯示，帶有FLP/Frt表現系統的新型桿狀病毒，可延長基因表現達到28天以上，並可有效促使ADSCs往硬骨細胞分化。我們將轉導過不同重組桿狀病毒的ADSCs混合種入PLGA載體並移植入紐西蘭白兔股骨缺陷(critical-sized femora bone defect)以評估加速骨缺陷修復的效果。X-ray結果顯示，實驗組(植入帶有FLP/Frt系統之ADSCs)不僅可於植入2週時間後，形成新骨連結骨缺陷；並且在植入4週時間後，所有動物皆完成骨缺陷修復(N>6)。另外藉由正子斷層(PET)，電腦斷層(CT)掃描，H&E染色與機械應力測試，亦發現與對照組(植入假性轉導或CMV啟動子表現之BMP2, VEGF)比較，混合移植經Bac-FCBW與Bac-FCVW轉導之ADSCs確實可加速大範圍骨缺陷修復。這些實驗結果顯示，結合FLP/Frt延長表現系統之新型桿狀病毒基因治療與ADSCs，可作為大範圍骨缺陷重建的另一選擇。

1. 文獻回顧 9
1-1 桿狀病毒 9
1-1-1 桿狀病毒之簡介 9
1-1-2 桿狀病毒之表現系統 9
1-1-2-1 昆蟲細胞之表現系統 10
1-1-2-2 桿狀病毒於哺乳動物細胞之表現系統 10
1-2 長效表現系統(FLP/FRT EXPRESSION SYSTEM) 12
1-2-1 長效表現生長因子 12
1-3 骨缺陷修復 12
1-3-1 複合生長因子對於骨修復之影響 13
1-3-2 利用桿狀病毒表現生長因子 14
1-3-3 利用骨隨幹細胞修復骨缺陷實驗 15
1-3-3-1 紐西蘭大白兔大腿骨骨缺損之簡介 15
1-4 脂肪幹細胞之簡介 15
1-4-1 脂肪幹細胞 15
1-4-2 脂肪幹細胞的誘導分化 17
1-5 實驗動機 19
2. 材料與方法 27
2-1 脂肪幹細胞純化 27
2-1-1 分離脂肪幹細胞 27
2-1-2繼代 27
2-2 重組桿狀病毒 28
2-2-1 建構重組桿狀病毒 28
2-2-2 轉染製作P0重組桿狀病毒 28
2-2-3 P1重組桿狀病毒 29
2-2-4 P2重組桿狀病毒 29
2-3 轉導策略 29
2-3-1 桿狀病毒病毒效價的測定 29
2-3-2轉導脂肪幹細胞 30
2-3-3間隔轉導脂肪幹細胞 30
2-4 流式細胞儀之分析 31
2-5 酵素免疫分析法 (ELISA) 31
2-6 鈣沉澱定量 32
2-7 紐西蘭大白兔長骨缺損修復實驗 33
2-7-1 紐西蘭大白兔長骨骨缺損之簡介 33
2-7-2 PLGA細胞支架製備 33
2-7-3 紐西蘭大白兔植入手術程序 34
2-8 骨缺陷修復評估 34
2-8-1 骨缺損修復初步評估 34
2-8-2 Critical defect的骨再生與血管重建評估 34
2-8-3 電腦斷層掃瞄(Computed Tomography Imaging，□CT) 35
2-8-4骨缺陷的代謝密度評估 35
2-8-5 H&E染色： 36
2-8-6 Critical defect血管新生評估： 36
2-8-7 critical defect的再生骨機械應力測試 36
3. 結果與討論 44
3-1 EGFP表現量 44
3-2 轉導方式與成效 45
3-2-1 轉導方式 46
3-2-2間隔轉導 47
3-2-3 延長表現 48
3-2-3 BMP-2表現量 49
3-2-4 VEGF表現量 49
3-3 分化程度 50
3-3-1 鈣沉澱量 50
3-4 股骨骨缺陷修復程度 51
3-4-1 X-ray 體外照相 52
4. 結論 62
5. 未來展望 64
5-1 免疫反應與安全性 64
5-1-1 免疫豁免 64
5-1-2 移植動物體內BMP-2之表現 64
5-1-3 移植動物產生抗人類BMP-2抗體(anti-hBMP-2 Ab)情形 64
5-2 HUVEC TUBULAR NETWORK FORMATION 65
6. 參考文獻 67

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