人骨形态发生蛋白-2基因腺病毒载体异位成骨

Ectopic bone formation using bone morphogenetic protein 2 adenoviral vector

目的:评价人骨形态发生蛋白2(hBMP-2)基因腺病毒表达载体(recombinant adenoviral vectorcarrying the human BMP-2 gene,AdBMP-2)成骨能力并探讨其成骨机制。方法:应用AdBMP-2体外感染兔骨髓间质干细胞(BMSC),7 d后观察hBMP-2和碱性磷酸酶(ALP)的表达,21 d后观察感染细胞钙结节形成能力。同时将AdBMP-2行裸鼠腓肠肌组织内注射,术后20 d取材,观察hBMP-2的表达及肌组织内异位成骨情况。结果:AdBMP-2可高效感染BMSC并获得hBMP-2的有效表达,ALP表达增高并形成钙结节。AdBMP-2在体内同样表达hBMP-2,并且以软骨化骨的方式启动成骨。结论:感染AdBMP-2的BMSC向成骨细胞分化,AdBMP-2在体内有着良好的成骨能力。

Objective To evaluate the osteoinductive activity of recombinant adenoviral vector carrying the human BMP-2 gene （AdBMP-2） and investigate the mechanism of bone formation induced by the bone morphogenetic protein 2 （BMP-2）. Methods AdBMP-2 infected rabbit bone messenchymal stem cells （BMSC） were cultivated in vitro. The expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis, and ALP was determined by enzyme cytochemistry 7 d after infection. At 21th day, Alizarin red S staining was used to evaluate calcium-rich deposits by cells transfected in culture. 30 μL AdBMP-2 solution （2 × 10^12 pfu · mL^-1 ） was injected into the muscle of the left hind limb of the nude rat, and 30 μL Adβ gal solution （2× 10^12pfu · mL^- 1） was injected into the muscle of the right hind limb as control. Animals were sacrificed at 20th day following injection to investigate the bone formation histologically and the expression of BMP-2 in the specimens of the injection sites by immunohistochemistry and in situ hybridization method. Results The expressions of BMP-2 and ALP in the transduced BMSC were confirmed at 7 th day after infection. A deeply stained mineralized layer was evident at 21th day in transduced cells. In vivo experiment, BMP-2 gene therapy induced mesenchymal monocytes chemotaxis, proliferation, differentiation into chondrocytes, with subsequent endochondral bone formation. At the same time,hBMP-2 expression was observed in the chondrocytes, osteoblasts and mesenchymal monocytes Conclusion AdBMP-2 posseses the osteoinductive activity in vitro and in vivo.