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

体内基因转移是最近开发的用于有效递送治疗性重组蛋白的装置。我们提出了一个假设，即高水平表达的骨形态发生蛋白-2（BMP-2）可能是一种未来的治疗方式，在体内诱导大量的骨形成。首先，将携带BMP-2基因的腺病毒直接注射到成年大鼠的比目鱼肌中来验证这一假设。通过腺病毒介导的转移，BMP-2基因在靶肌肉中成功地过表达，而在这种情况下，肌肉内和周围的骨形成未能发生。其次，为了募集假定的骨祖细胞，我们通过原位移植同时进行基因转移来诱导靶肌肉的缺血性变性。BMP-2基因转移和原位肌肉移植的组合导致几乎整个移植肌肉的成功骨化，而无论是单独的肌肉移植还是肌肉移植和腺病毒介导的报告基因转移的组合，LacZ诱导肌肉中的任何骨形成。通过组织切片的阳性von Kossa染色和该区域的X线不透性，骨化过程是明显的。研究还发现，在移植肌肉中过表达的BMP-2转基因抑制了肌肉再生，否则肌肉再生将跟随肌肉变性。我们进一步证实了BMP受体IA型在移植肌肉中的上调，表明其参与骨形成过程。总之，BMP-2基因的过度表达诱导了移植物诱导的缺血性变性下骨骼肌的大量异位骨化，这可能上调了原位骨祖细胞。

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.