hVEGF_(165)及hBMP-7共表达重组腺相关病毒载体体内诱导成骨及成血管研究

STUDY ON EFFECT OF RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR CO-EXPRESSING HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR 165 AND HUMAN BONE MORPHOGENETIC PROTEIN 7 GENES ON BONE REGENERATION AND ANGIOPOIESIS IN VIVO

目的探讨hVEGF165及hBMP-7共表达重组腺相关病毒(recombinant adeno-associated virus,rAAV)体内诱导成骨及成血管的活性,为股骨头缺血性坏死(avascular necrosis of the femoral head,ANFH)的AAV基因治疗提供理论基础。方法取体外培养的第3代兔BMSCs,分别采用以下4种病毒转染并分为4组:rAAV-hVEGF165-内部核糖体进入位点序列(internal ribosome entry site,IRES)-hBMP-7(A组)、rAAV-hVEGF165-绿色荧光蛋白(green fluorescent protein,GFP)(B组)、rAAV-hBMP-7-GFP(C组)及rAAV-IRES-GFP(D组)。取24只雄性新西兰大耳白兔制作后肢缺血模型,并随机分为4组(n=6),于股骨肌肉内植入上述4组病毒转染的BMSCs,8周后应用超声影像学检测兔后肢胫前动脉血流量,行CD34免疫组织化学染色检测微血管生成。另取24只雄性新西兰大耳白兔制作股骨肌袋模型,并随机分为4组(n=6),于肌袋内植入上述4组病毒转染的BMSCs,8周后应用X线片检测兔后肢原位骨化,行von Kossa钙盐染色检测肌肉组织成骨矿化。结果病毒注射后动物未出现局部或全身毒性反应。病毒注射后8周,A组兔后肢胫前动脉血流百分比及CD34阳性微血管数均高于其他3组,差异有统计学意义(P<0.05);B组高于C、D组,差异有统计学意义(P<0.05);C、D组间比较差异无统计学意义(P>0.05)。病毒注射后8周,A、C组兔后肢肌袋内出现可被X线检测到的原位骨化,von Kossa肌肉组织钙盐染色可见大量钙盐沉积,且A组原位骨化及钙盐沉积程度均较C组强;B组及D组均未见明显原位骨化及钙盐沉积形成。结论 rAAV-hVEGF165-IRES-hBMP-7载体具有体内诱导成骨及成血管的生物学活性。

Objective To study the effect of recombinant adeno-associated virus （rAAV） vector co-expressing human vascular endothelial growth factor 165 （hVEGF165） and human bone morphogenetic protein 7 （hBMP-7） genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of the femoral head （ANFH）. Methods Twenty-four male adult New Zealand rabbits were made the ischemic hind limb model and divided into 4 groups （n=6）. The 3rd generation rabbit bone marrow mesenchymal stem cells （BMSCs） were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively： rAAV-hVEGF165-internal ribosome entry site （IRES）-hBMP-7 （group A）, rAAV-hVEGF165-green fluorescent protein （GFP） （group B）, rAAV-hBMP-7-GFP （group C）, and rAAV-IRES-GFP （group D）. At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind limb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the proliferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups （n=6）. The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineralization. Results No symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups （P 0.05）. The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D （P 0.05）. However, there was no significant difference between group C and group D （P 0.05）. At 8 weeks after injection, orthotopic ossification and mineralization were evidently detected in group A and group C, and group A was stronger than group C. No obvious evidence of orthotopic ossification and mineralization were observed in group B and group D. Conclusion rAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.