桿狀病毒結合長效表現系統轉導紐西蘭大白兔的間葉幹細胞並應用在頭蓋骨缺陷的修復

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。桿狀病毒轉導外源基因表現，雖然有相對於其他病毒載體安全的優勢，但是其最大缺點就是表現時間太短，對於需要長時間表現外源治療性基因應用時，其效果不佳。因此，我們以新建構的FLP/Frt hybrid重組桿狀病毒，媒介較長時間的生長因子表現，以達較佳的骨缺陷修復效果。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同種異體紐西蘭大白兔之間葉幹細胞後是否可以有效地修復大範圍頭蓋骨缺陷。首先我們利用帶有FLP/Frt系統的基因重組桿狀病毒Bac-FCOEE決定轉導間隔時間與轉導病毒劑量，並利用d2EGFP結合FLP/Frt與WPRE分析基因延長表現，之後以 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))轉導紐西蘭大白兔之間葉幹細胞，觀察頭蓋骨缺陷修復效果。結果顯示，在病毒劑量同為MOI 100的Bac-FLP和帶有Frt site的重組桿狀病毒在間隔4小時進行連續轉導，其重組效率最佳，且並無明顯抗病毒反應，讓帶有FLP/Frt表現系統的新型桿狀病毒可延長基因表現可達到28天。我們將轉導過Bac-FCBW和Bac-FCVW的BMSCs混合種入PLGA載體並移植入紐西蘭白兔頭蓋骨缺陷(critical-sized calvarial bone defect)以評估加速骨缺陷修復的效果。X-ray與H&E染色結果顯示，mock與Bac-CB/Bac-CV的骨缺陷修復情形並不佳，而Bac-FCBW/Bac-FCVW實驗組在植入12週後，確實可以修復缺陷。

關鍵字

桿狀病毒； BMP-2 ； VEGF ；骨髓幹細胞； FLP/Frt

並列摘要

A segmental bone fracture generally necessitates extended and complicated surgical methods related to bone and vessel reconstruction, thus autologous or allogenic bone grafting is usually required.Recently, mesenchymal stem cells (MSCs)-based cell therapy and virus-based gene therapy have converged and have shown great potential in assisting and accelerating bone healing. Baculovirus is a novel vector for gene delivery into stem cells, but it only mediates transient expression. Therefore we developed a long-term system whereby one baculovirus expressed FLP recombinase (Bac-FLP) while the other hybrid baculovirus harbored an Frt flanking transgene cassette to assist and accelerate segmental bone healing. In this study, we explored whether FLP/Frt-hybrid baculovirus vector engineered rabbit MSC are able to assist and accelerate segmental calvarial bone healing. Within the BMSCs transduced with Bac-FLP and Bac-FCOEE, all subsequent transductions were performed at MOI 100/100 at an interval of 4 h and no obvious anti-virus reaction, enabling transgene persistence in episomal form and prolonging the expression to >28 days. BMSCs engineered by the conventional baculovirus transiently expressing BMP2/VEGF (S group) only healed the critical size (8 mm) segmental calvarial bone defects in 20 % of New Zealand White rabbits at 12 weekspost-implantation, whereas BMSCs engineered by the hybrid vectors persistently expressing BMP2/VEGF (L group) healed the critical-size defects in 87 % in 8 weeks. Compared with the S group, the L group can accelerated the healing

並列關鍵字

baculovirus ； BMP-2 ； VEGF ； BMSC ； FLP/Frt

參考文獻

Airenne KJ, Hiltunen MO, Turunen MP, Turunen AM, Laitinen OH, Kulomaa MS, Yla-Herttuala S. 2000. Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery. Gene Therap 7(17):1499-504.

Bilello JP, Cable EE, Myers RL, Isom HC. 2003. Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatopytes. Gene Therapy 10(9):733-749.

Boyce FM, Bucher NL. 1996. Baculovirus-mediated gene transfer into mammalian cells. Proceedings of the National Academy of Science 93(6):2348-52.

Bruder SP, Jaiswal N, Haynesworth SE. 1997. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. Journal of Cellular Biochemistry 64(2):278-294.

Bruder SP, Kurth AA, Shea M, Hayes WC, Jaiswal N, Kadiyala S. 1998. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. Journal of Orthopaedic Research 16(2):155-162.

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桿狀病毒結合長效表現系統轉導紐西蘭大白兔的間葉幹細胞並應用在頭蓋骨缺陷的修復

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