Osteogenesis induced by the new drug delivery system, in vivo gene transfer

体内基因转移是最近开发的一种有效传递治疗性重组蛋白的装置。我们提出了一个假设，即骨形态发生蛋白2 （BMP-2）的高水平表达可能是未来在体内诱导大量骨形成的治疗方式。为了验证这一假设，首先将携带BMP-2基因的腺病毒直接注射到成年大鼠比目鱼肌中。通过腺病毒介导的转移，BMP-2基因成功地在靶肌肉中过表达，而在这种情况下，肌肉内部和周围的骨形成未能发生。其次，为了招募假定的骨祖细胞，我们然后通过将其与基因转移同时原位移植来诱导目标肌肉的缺血性变性。BMP-2基因移植与原位肌肉移植的结合几乎可以使整块移植肌肉成功成骨，而无论是单纯的肌肉移植，还是肌肉移植与腺病毒介导的报告基因转移的结合，LacZ都不能诱导肌肉成骨。组织切片von Kossa染色呈阳性，该区域放射不透明，可见骨化过程。我们还发现移植物肌肉中BMP-2基因的过表达抑制了肌肉的再生，否则肌肉就会发生变性。我们进一步证明了移植肌肉中BMP受体IA型的上调，表明其参与骨形成过程。综上所述，BMP-2基因的过表达诱导了移植物缺血性变性下骨骼肌的大量异位骨化，其可能是原位骨祖细胞的上调。

In vivo gene transfer is a recently-developed device for efficient delivery of a therapeutic recombinant protein. We formulated the hypothesis that a high level of expression of bone morphogenetic protein-2 (BMP-2) could be a future therapeutical modality in terms of inducing substantial bone formation in vivo. First to test this hypothesis, adenoviruses carrying BMP-2 gene were directly injected into the soleus muscle of adult rat. The BMP-2 gene was successfully overexpressed in the target muscle by adenovirus-mediated transfer, whereas bone formation in and around the muscle failed to occur in this case. Secondly, in order to recruit putative osteoprogenitor cells, we then induced ischemic degeneration of the target muscle by orthotopically grafting it simultaneously with the gene transfer. The combination of BMP-2 gene transfer and orthotopic muscle grafting resulted in successful ossification of almost the whole grafted muscle, whereas neither muscle grafting alone nor the combination of muscle grafting and adenovirus-mediated transfer of reporter gene, LacZ induced any bone formation in the muscle. The ossification process was evident by positive von Kossa staining of the histological sections and roentogenographical radio-opacity of the region. It was also found that the BMP-2 transgene overexpressed in grafted muscles inhibited muscle regeneration, which should otherwise follow the muscle degeneration. We further demonstrated an upregulation of BMP receptor type IA in grafted muscles, suggesting its involvement in the bone formation process. In conclusion, overexpression of BMP-2 gene induced massive heterotopic ossification in skeletal muscles under graft-induced ischemic degeneration, which possibly upregulates osteoprogenitor cells in situ.