TGF-β信号通路调控骨膜干细胞参与小鼠骨折修复的分子机制及补肾活血方的干预研究

Fracture is a relatively common disease in orthopedics and the rate of delayed healing or nonunion is still about 5-10%. Bushenhuoxue formula is an effective treatment for bone fracture in clinic while its mechanism of action is not completely known. Recently, some researchers identified that periosteal stem cells (PSCs) efficiently contribute to bone formation and bone regeneration. But, the exact mechanism of the PSCs in bone repair is somewhat unclear. TGF-β signaling which promotes stem cell migration and differentiation is very likely a pivotal signaling in fracture repair. Our pre-experiment showed that Bushenhuoxue formula drug serum accelerated the migration of mesenchymal stem cells via TGF-β signaling. Hence, we hypothesize that Bushenhuoxue formula accelerates fracture repair by regulating periosteal stem cells via TGF-β signaling. In this research, we intend to perform tibial fracture procedure in tamoxifen-mediated lineage tracing of PSCs in Gli1CreErt2; tdTomato mice which could allow us to observe the PSCs during bone repair process and trace the molecular processes in vivo. In the meantime, we resort to TGF-β antibody and Gli1CreErt2;TGFβRIIflox/flox conditional knockout transgenic mice to thoroughly investigate the effects of TGF-β signaling on proliferation, migration and differentiation of PSCs. Findings in this research will clarify the mechanism of PSCs in fracture repair and provide sufficient evidence for treatment with Bushenhuoxue herbs in bone fracture.

骨折是骨科常见疾病之一，约5-10%患者存在骨折延迟愈合或不愈合。补肾活血方治疗骨折具有较好疗效，其作用机制值得进一步研究。近年来，研究表明骨膜干细胞（Periosteal stem cells，PSCs）参与骨形成及骨折修复过程，但其具体机制目前仍不明确，而TGF-β信号通路具有促进干细胞迁移分化作用。本项目组通过前期实验发现补肾活血方含药血清经TGF-β信号通路促进间充质干细胞迁移，因此提出科学假说：“补肾活血方介导TGF-β信号通路调控骨膜干细胞促进骨折愈合”。本研究拟利用细胞体内示踪技术构建PSCs报告小鼠，建立骨折模型，对骨折修复过程中的PSCs定向示踪，同时借助TGF-β中和剂及PSCs特异性TGFβRII基因敲除报告小鼠，探究TGF-β信号通路对PSCs增殖、迁移及分化影响。研究结果将进一步明确PSCs参与骨折修复的作用机制以及为补肾活血中药促进骨折修复提供充分的实验依据。

骨膜在骨折愈合过程中非常重要，骨膜缺失导致骨折愈合受损甚至骨折不愈合。然而，骨膜内的细胞鉴别还不清楚。与传统的体外细胞行为鉴定不同，体内谱系追踪实验实现了在未受干扰的人体内鉴定骨膜祖细胞。在本研究中，通过分析TomatoGli1ER在谱系追踪转基因小鼠中，我们发现了Gli1+细胞是长时间驻留在长骨骨膜内的细胞。有趣的是，Gli1 +幼鼠骨膜细胞及其后代大量存在，但7月龄时明显减少。我们的骨膜去除实验证明Gli1+细胞对骨折愈合的作用。通过追踪Gli1+细胞的命运，我们发现Gli1+骨膜细胞作为祖细胞在体内自我更新和多向分化，对骨痂形成和骨折修复有重要作用。IF分析显示，TGF-β1在骨痂微环境中大量富集。局部注射TGF-β中和剂抑制Gli1+骨膜细胞TGF-β信号的研究，可见骨膜扩增及后续的软骨和骨痂组织明显减少，导致骨愈合延迟和软骨内骨形成受损。证实了TGF-β/的重要作用Smad2信号在Gli1+调控中的作用骨膜细胞增殖分化和骨折愈合。中药经典方BSHXF在临床实践中，对骨折患者的治疗效果显著。我们发现BSHXF增强小鼠骨折修复。包括X线和显微CT分析表明，BSHXF能促进骨痂的形成，加速骨愈合。我们的研究提示TGF-β/Smad信号通路在调节骨折愈合过程中起关键作用。