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研究生:林進裕
研究生(外文):Lin, Chin-Yu
論文名稱:以桿狀病毒工程改造間葉幹細胞修復紐西蘭白兔股骨典型範圍缺陷
論文名稱(外文):Baculovirus-Engineered Mesenchymal Stem Cells Heal the Critical-Sized Femoral Segmental Bone Defects in Rabbits
指導教授:胡育誠胡育誠引用關係
指導教授(外文):Hu, Yu-Chen
口試委員:閻紫宸楊台鴻張鑑中胡育誠張毓翰
口試日期:2011-07-12
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:156
中文關鍵詞:桿狀病毒組織工程骨缺陷修復基因治療
外文關鍵詞:baculovirustissue engineeringbone defect regenerationgene therapy
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  • 收藏至我的研究室書目清單書目收藏:1
大範圍的骨缺陷通常發生於外傷或進行癌症外科手術切除之後,造成許多外觀缺陷與行動上的不便,據統計在美國每年花費150億美金以上於骨修復相關的治療。本研究首先探討桿狀病毒工程改造之骨髓間葉幹細胞(bone marrow derived stem cells, BMSCs)是否可以修復大範圍骨缺陷。實驗以表現第二型骨型態蛋白(BMP2)或血管內皮細胞生長因子(VEGF)之重組桿狀病毒轉導紐西蘭兔BMSCs,並共同移植入紐西蘭白兔骨股典型範圍缺陷(10 mm)。X-ray結果顯示,經桿狀病毒工程改造之BMSCs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,所有實驗動物皆完成骨缺陷修復。由正子斷層掃描、電腦斷層掃描、組織切片化學染色與機械應力測試分析,亦發現與其他對照組比較,共同植入表現BMP2與VEGF的BMSCs可顯著促進骨修復與應力承受特性,其骨修復的同時亦伴隨著旺盛的血管新生。此外,從脂肪分離幹細胞(adipose derived stem cells, ASCs)比從骨髓分離幹細胞容易,但是曾有文獻報導ASCs無法使用於股骨大面積缺損之修復。我們假設若能延長生長因子表現,可能可以進一步改善ASCs應用於硬骨的修復。因此我們建構一新型的混成型長效桿狀病毒,證實在轉導ASCs後可切割重組桿狀病毒基因體形成細胞內質體,並延長轉殖基因表現時間至28天以上。此外,應用此混成型桿狀病毒改質ASCs可成功修復紐西蘭兔股骨大面積缺損。相較於短暫表現BMP2/VEGF的組別,於ASCs移植後12週僅完成40%動物股骨缺損修補。長效表現BMP2/VEGF的組別,於術後8週,可100%完成動物股骨缺損修補。最後,我們更進一步探討免疫細胞(如巨噬細胞、CD4+、CD8+ T cells)浸潤移植部位的情形,以及週邊血液淋巴球細胞、脾臟細胞對桿狀病毒轉導之ASCs的免疫反應。確認以混成型桿狀病毒改質之ASCs,在移植紐西蘭兔股骨缺損後,不會誘發嚴重之免疫排斥與產生抗原專一性細胞免疫反應。本研究釐清經由桿狀病毒轉導及轉殖基因表現後,ASCs的免疫特性是否有所改變,對於桿狀病毒是否能成為主流基因載體非常重要。
Massive segmental bony defects often occur following trauma or tumor resection which may result in non-union and even physical impairment, and over 15 billion dollars are spent annually in the US for the bone regeneration related therapies. Recently, mesenchymal stem cells (MSCs)-based therapy and virus-based gene therapy have converged and hold promise in assisting and accelerating bone healing. To explore whether BMSCs engineered by baculovirus can heal large bone defects, the New Zealand White (NZW) rabbit BMSCs were transduced with the BMP2-expressing baculovirus or VEGF-expressing baculovirus, co-seeded to scaffolds and co-implanted into critical-sized (10 mm) femoral segmental defects in NZW rabbits. X-ray analysis revealed that the baculovirus-engineered BMSCs not only bridged the defects at as early as week 2, but also healed the defects in 100% of rabbits at week 4. When compared with other control groups, the BMP2/VEGF-expressing BMSCs remarkably enhanced the segmental bone repair and mechanical properties, as evidenced by positron emission tomography (PET), micro-computed tomography (?媴T), histochemical staining and biomechanical testing. The immunohistochemical staining further attested that the ameliorated bone healing concurred with the augmented angiogenesis. Additionally, adipose-derived stem cells (ASCs) also hold great promise for tissue regeneration because, unlike BMSCs requiring bone marrow harvest, ASCs are easy to isolate through liposuction and are more applicable in the future clinical application. But, ASCs were reported to failed in femoral segmental bone defect healing. To solve this problem, we hypothesized that sustained expression of factors promoting osteogenesis (BMP2) and angiogenesis (VEGF) in ASCs may provide continuous stimuli to augment the bone healing. Therefore we developed a hybrid baculovirus system and attested the ASCs transduced with the hybrid baculovirus can leading to cassette excision off the baculovirus genome, enabling transgene persistence in episomal form and prolonging the expression to >28 days. Compared with the ASCs engineered by the conventional baculovirus transiently expressing BMP2/VEGF only healed the critical-size segmental femoral bone defects in 40% of rabbits at 12 weeks post-implantation, whereas ASCs engineered by the hybrid vectors persistently expressing BMP2/VEGF healed the critical-size defects in 100% of animals in 8 weeks. Thereby attesting our hypothesis that persistent BMP2/VEGF expression is essential in use of ASCs to treating massive segmental defects necessitating sustained stimuli. Moreover, the in vivo immunological evaluations are evaluated after the hybrid baculovirus-transduced ASCs are implanted into the femoral critical defects. The activation of immune cells (e.g. macrophage and T cells) are observed by immuno histological staining and the recipient lymphocytes against donor-ASCs are evaluated by spleen derived mixed lymphocyte cytotoxic assays and peripheral blood differential count. These data demonstrated that the transplantation of ASCs engineered by hybrid baculovirus will not elicit severe immune rejection and antigen specific cellular immune response. These in vivo experiments will shed light on whether baculovirus transduction and transgene expression trigger unwanted immune responses and change immunocharacteristics of ASCs in vivo, which will benefit future baculovirus-mediated gene therapy.
第一章 序論 1
第二章 文獻回顧 3
2-1 骨組織工程簡介 3
2-1-1骨骼系統之重要性與常見傷害 3
2-1-2 骨骼的組織與組成 4
2-1-3 骨細胞與骨骼重建機制 5
2-1-4 骨組織工程的發展 7
2-2 幹細胞的特性與在組織工程上之應用 8
2-3 基因治療 9
2-4 桿狀病毒表現系統 11
2-4-1 桿狀病毒之特性 11
2-4-2 桿狀病毒應用於組織工程 13
2-4-3 FLP/Frt重組系統 14
2-5 研究動機 15
第三章 材料與方法 22
3-1 重組桿狀病毒之建構與製備 22
3-1-1 昆蟲細胞培養 22
3-1-2 表現載體(donor plasmid)之建構 22
3-1-3 重組表現載體之轉置反應(transposition) (Bac-to-Bac system) 24
3-1-4 重組bacmid之分離 24
3-1-5 重組bacmid之轉染反應(transfection) (製備p0病毒) 25
3-1-6 基因重組桿狀病毒放大培養 25
3-1-7 病毒感染力價之定量(infectious titer) 26
3-2 紐西蘭白兔幹細胞分離培養 27
3-2-1紐西蘭白兔BMSCs分離培養 27
3-2-2 紐西蘭白兔ASCs分離培養 28
3-3 紐西蘭白兔BMSCs與ASCs之轉導(transduction) 29
3-3-1 基因重組桿狀病毒之轉導 29
3-3-2 流式細胞儀之分析 29
3-4 酵素免疫分析(ELISA)生長因子之含量 30
3-5基因重組桿狀病毒轉導BMSCs或ASCs後體外分化測試 31
3-5-1 Osteopontin 基因表現(qRT-PCR) 31
3-5-2 Alkaline phosphatase (ALP)表現分析 33
3-5-3 茜紅素(alizarin red)染色分析 34
3-5-4 鈣沉積定量 35
3-5-5 Bac-CV轉導BMSCs之培養液刺激血管新生 35
3-6 細胞載體製備與測試 37
3-6-1 PLGA載體製備 37
3-6-2 3D培養生長因子分泌測試 37
3-6-3動物試驗所需之載體製備 38
3-7 紐西蘭白兔股骨critical defect 模型建立與載體植入程序 39
3-8 Non-invasive骨缺損修復評估 40
3-8-1 X-ray攝影評估骨缺陷修復情況 40
3-8-2 正子斷層掃描評估骨缺陷位置代謝情況 40
3-9 Critical defect的骨再生與血管重建評估 41
3-9-1 電腦斷層掃瞄(Computed Tomography, CT) 41
3-9-2 H&E染色 42
3-9-3 Critical defect血管新生評估-組織切片螢光免疫染色 42
3-10 Critical defect位置新生骨機械應力測試 43
3-11 紐西蘭兔脾臟細胞分離 43
3-12 混合型淋巴細胞毒殺試驗 45
3-13 統計學分析 46
第四章 結果與討論(I) 52
以桿狀病毒工程改質骨髓來源之幹細胞修復紐西蘭兔股骨典型範圍缺陷
4-1 基因重組桿狀病毒轉導紐西蘭白兔BMSCs效率 52
4-2 以Bac-CB轉導BMSCs並誘導往硬骨細胞分化 52
4-3 以Bac-CV轉導BMSCs並分析刺激血管新生能力 54
4-4 紐西蘭兔股骨缺陷動物模式與X-ray評估骨缺陷修復 55
4-5 骨缺陷修復過程中骨代謝活性分析 56
4-6 股骨修復情況與電腦斷層掃描分析 57
4-7 組織切片染色分析骨組織新生與血管新生 58
4-8 機械扭轉測試股骨修復情況 59
4-9 討論 59
第五章 結果與討論(II) 76
以新型長效表現型桿狀病毒工程改質ASCs修復紐西蘭兔股骨典型範圍缺陷
5-1 FLP/Frt系統媒介桿狀病毒基因體重組最佳化條件測試 77
5-2應用FLP/Frt系統之新型桿狀病毒延長轉殖基因表現 79
5-3以X-ray攝影方式評估活體動物股骨修復情況 81
5-4以PET/CT觀察活體動物骨組織代謝活性 81
5-5動物犧牲後的新生骨外觀及?媴T評估 82
5-6組織學分析骨再生情形 83
5-7扭轉機械應力測試新生骨品質 84
5-8 討論 84
第六章 結果與討論(III) 96
長效型桿狀病毒工程改質ASCs修復股骨典型範圍缺陷之效率與免疫特性探討
6-1移植後採周邊血液分析血球分類計數 97
6-2移植後採周邊血液分析重組蛋白表現 99
6-3組織移植處之發炎反應觀察 100
6-4組織移植處之免疫細胞浸潤 101
6-5混合型淋巴細胞分析其抗原專一性 102
6-6 討論 103
第七章 結論與未來展望 112
7-1 移植動物產生抗人類BMP2 / VEGF抗體分析 113
7-2 同種異體移植間葉幹細胞追蹤 113
7-2-1 報導基因建構與穩定表現報導基因的幹細胞株選植 114
7-2-2 以SPECT分析骨缺陷修復動物內同種異體移植MSC量 114
7-2-3 組織切片分析移植幹細胞在動物體內的分布 115
7-3 較長術後時間觀察骨修復品質與組織癌化評估 115
7-3-1 電腦斷層掃描分析 116
7-3-2 新生骨組織生物力學測試 116
7-3-3 新生骨組織重整(bone remodeling)與骨吸收(bone resorption) 117
7-3-4 新生骨組織癌化評估 117
第八章 參考文獻 120
第九章 附件 130
9-1 參加研討會論文摘要 130
9-2 已發表於國際期刊之論文 138

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