肌肉影响骨折愈合的Akt1信号通路分子机制研究

It has been demonstrated that muscle and soft-tissue injury is inevitable in fracture or during its treatment process, which might lead to the reversible or permanent muscle atrophy. By using our unique animal model of reversible muscle atrophy, we have proved that muscle atrophy could significantly delay fracture healing. But until now, the mechanism about how muscle atrophy affects fracture healing and how the mechanical signals of muscle converts into biological signals is not clear. Therefore, we intend to use cell culture technique to explore the influence of cyclic mechanical tensile force/pressure on the functions of myoblasts, mesenchymal stem cells, osteoblasts and osteoclasts in vitro. And then deep correlation between cell Akt1 signaling pathway and cyclic mechanical tensile force/pressure will be verified. In vivo, we will dynamically observe the changes of protein level and gene expression of Akt1 signaling pathway in muscles and fracture fragments and the functional changes of mesenchymal stem cells, osteoblasts and osteoclasts in the process of muscle atrophy and recovery over time. Furtherly, we are going to clarify the effect and mechanism of Akt1 signaling pathway on fracture healing. The antagonistic effect of Akt1 signaling pathway on muscle atrophy and its regulation on fracture healing will be ascertained by its targeted activation. The completion of this study will provide a new idea method for the targeted treatment of fracture. The ultimate aim of this study is to improve the clinical efficacy of fracture.

业已证实，骨折的发生和治疗过程中，不可避免地会发生肌肉与软组织的损伤，从而导致肌肉发生可逆性或永久性的萎缩。借助我们所独创的可逆性肌肉萎缩实验模型完成的研究表明，骨折端周围肌肉萎缩可明显延缓骨折愈合。但肌肉萎缩及相应的肌肉的力学信号改变如何转换成生物学信号，进而影响骨折愈合，其机制尚未明确。为此，我们拟借助体外细胞培养平台，探明周期性连续拉伸力/压力刺激对肌细胞和干细胞、成骨及破骨细胞功能的影响，及与Akt1信号通路的相关性；应用可逆性肌肉萎缩实验模型，在肌肉萎缩及随时间恢复的过程中，动态观察肌肉与骨折部位Akt1信号通路相关蛋白和基因表达水平的改变，及干细胞、成骨和破骨细胞功能的变化，阐明Akt1信号通路在肌肉影响骨折愈合中的作用及机制；靶向激活Akt1信号通路，观察其对拮抗肌肉发生萎缩及调控骨折愈合的作用。研究的完成将为骨折治疗提供一种崭新的思路。本研究的最终目标是提高骨折的临床疗效。

肌肉损伤及废用性肌肉萎缩均不利于骨折愈合。文献报道Akt信号通路不仅可以通过干预成骨细胞及破骨细胞的生物学行为参与骨代谢的调节，还在骨骼肌的萎缩及肥大过程中扮演重要角色。本研究的目的是结合课题组前期独创的大鼠骨折-肌萎缩模型，即采用肉毒素肌肉注射造成大鼠股四头肌可逆性肌萎缩，1周后再完成股骨骨折造模，在骨折愈合过程中中靶向调节Akt信号通路，研究其对肌肉萎缩恢复及骨折愈合的作用，并探讨肌肉影响骨折愈合的相关机制。在肌萎缩恢复伴骨折愈合过程中，应用Akt信号特异性小分子激动剂SC79 (20毫克/公斤/周)或抑制剂MK2206(100毫克/公斤/周)靶向调节Akt信号，观察肌肉萎缩的恢复及骨折愈合情况。我们发现SC79可以显著加速萎缩的股四头肌重量和肌纤维大小的恢复。此外，SC79上调Akt信号通路后可以有效地促进骨痂形成，并提升骨痂的力学强度。免疫组化结果显示，SC79上调Akt信号通路后不仅可以促进萎缩的肌肉表达肌球蛋白重链，同时抑制萎缩肌肉表达、分泌肌骨素，通过调节骨折端的应力刺激及干预骨髓间充质干细胞的成骨分化，最终促进骨折愈合。体外细胞学实验亦证实SC79可以增加L6成肌细胞在成肌分化过程中MyoD、MRF4、Myf5及MHC1/10的基因表达，并促进肌管形成。综上，研究发现SC79上调Akt信号通路可以通过加速萎缩肌肉的恢复进而促进骨折愈合，为临床上骨折伴肌萎缩的治疗提供了一种新的思路。