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 (□CT), 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.

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