Rspo1-LGR4轴调控辐射后BMSC命运维持骨稳态的作用及机制

In an effort to explore the underlying mechanisms by which BMSC maintains bone homeostasis under radiation microenvironment, we have recently found that radiation impairs the self-renewal ability and osteogenic differentiation potential of BMSC while enhances adipogenesis, along with an increased expression of Rspo1 and LGR4 receptor. Of note, knockdown of Rspo1 or LGR4 worsens the deleterious effects of radiation on BMSC. External Rspo1 abolishes radiation-induced activation of mTOR signaling and inhibition of autophagy of BMSC. Besides, administration of Rspo1 recombinant protein alleviates radiation-induced bone loss and marrow adiposity. Such information leads to the novel hypothesis that autocrine of Rspo1-LGR4-mTOR-autophagy axis may act as key mechanisms by which BMSC exerts radio-protective role on itself as well as bone tissue. Thus in this grant, LGR4 conditional knockout mice are supposed to be applied to reversely prove that the Rspo1-LGR4 signaling is an essential means by which BMSC orchestrates bone homeostasis after radiation. Further, we aim to reveal the effects of Rspo1-LGR4 axis on fate determination of irradiated BMSC. Finally, the relationship of Rspo1-LGR4 axis to mTOR-autophagy signaling to exert radio-protective role on BMSC and bone tissue will also be elucidated. By doing so, we hope to provide new drug targets for the treatment of radiation-induced bone injury.

我们在探讨骨髓间充质干细胞（BMSC）维持辐射微环境下骨稳态机制的过程中，发现BMSC辐射后，其干性削弱、成骨分化减弱而成脂分化增强，同时伴有Rspo1及其受体LGR4表达上调；如沉默Rspo1或LGR4，将加剧辐射后BMSC干性与分化表型的改变，而外源性Rspo1可减少辐射所致的BMSC mTOR信号通路激活，增加BMSC的自噬，缓解骨量丧失和骨髓脂肪化。据此我们提出：自分泌Rspo1-LGR4-mTOR-自噬作用轴，可能是辐射后BMSC启动防御修复、维持骨稳态的重要机制。为此，本项目拟采用LGR4间充质细胞条件敲除小鼠，进一步反向佐证Rspo1-LGR4轴在BMSC维持辐射后骨稳态中的关键作用；继而探索该信号轴对辐射后BMSC命运的调控模式；最后联合体内外研究，验证mTOR-自噬通路在Rspo1-LGR4轴介导的BMSC及骨组织放射保护中的作用，为建立放射骨损伤防治新策略提供科学依据。

骨组织放射损伤是肿瘤放疗后的常见并发症。近年来，移植自体或同种异体来源的间充质干细胞（Mesenchymal stem cell, MSC）在机体放射损伤修复中的作用已经得到广泛验证。然而迄今为止，关于骨组织原位的MSC（BMSC）在维持辐射后骨稳态中的作用及其机制仍不清楚。. 本课题通过研究R-spondin1/LGR4对骨组织及骨髓间充质干细胞的放射保护作用，就R-spondin1对小鼠放射性骨损伤的干预作用进行了初步体内探索，同时验证辐射微环境下R-spondin1/LGR4对BMSC自我更新能力及成骨、成脂向分化潜能的影响。最后，运用生物信息学分析介导R-spondin1/LGR4对BMSC的放射保护作用的可能机制，在此基础上阐述了mTOR-自噬通路在上述过程中的作用。主要研究结果和结论如下：.1. 通过小鼠胫骨放射损伤模型，发现辐射导致骨量减少，骨髓腔脂肪化；R-spondin1对辐射微环境下的骨量具有正向调节作用，此外，抑制辐射引起的骨髓脂肪化，对骨组织起放射保护作用。.2. 4Gy辐射导致BMSC DNA损伤，削弱BMSC的干性，抑制BMSC成骨向分化，促进其成脂向分化；R-spondin1可以通过LGR4减少辐射微环境下BMSC的DNA损伤，维持BMSC的自我更新能力，促进其成骨向分化，抑制其成脂向分化；.3. 通过RNA Sequence，发现mTOR-自噬通路在介导辐射引起的BMSC损伤，以及R-spondin1/LGR4对BMSC的放射保护中可能起重要作用。进一步实验证明，辐射微环境下BMSC自噬水平降低，R-spondin1/LGR4可以通过抑制mTOR通路激活细胞自噬，从而维持BMSC的自我更新能力及成骨向分化潜能，抑制其成脂向分化作用。. 以上努力将为多维度了解放射性骨保护新机制、建立放射性骨损伤防治新策略提供理论与实验依据。