ESCRT调节的细胞自噬在肥大软骨细胞命运决定中的机制研究

Conventional wisdom of endochondral ossification has been greatly challenged by the findings of multiple fates of hypertrophic chondrocytes. However, the cellular and molecular mechanisms underlying the fate determination of hypertrophic chondrocytes remain completely unknown. Lines of clues suggest that autophagy is tightly associated with the fate of hypertrophic chondrocytes. Functions of endosomal sorting complex required for transport (ESCRT) complexes are involved in control of autophagy by multivesicular bodies formation. Our previous studies have established multiple genetic mouse models in which the key component of ESCRT complexes Hgs was deleted specifically in growth plate chondrocytes and hypertrophic chondrocytes. Interestingly, the endochondral ossification as well as the conversion of hypertrophic chondrocytes into other types of stromal cells were greatly hampered in mutant mice. In this study, we will employ these unique mouse models to in-depth study the mechanisms underlying the essential role of ESCRT-regulated autophagy in fate determination of hypertrophic chondrocytes, and thus conceptually renew our knowledge of endochondral ossification.

肥大软骨细胞转分化现象的发现改写了人们对软骨内成骨的认识，然而决定肥大软骨细胞命运的细胞和分子机制一无所知。种种迹象提示细胞自噬与肥大软骨细胞的命运关系密切。转运必需内体分选复合物（ESCRT）通过多囊泡体的形成参与细胞自噬。我们前期的研究构建了在软骨细胞和肥大软骨细胞中特异性敲除ESCRT复合体重要组分Hgs的小鼠模型，发现基因敲除小鼠软骨内成骨异常，肥大软骨细胞向其他类型基质细胞的转变受到了明显的阻滞。本研究拟利用多种基于Cre-LoxP系统建立的细胞谱系示踪及基因功能验证体系，深入研究ESCRT调节的自噬在肥大软骨细胞命运决定中的机制，进而更新我们对软骨内成骨的理解和认识。

肥大型软骨细胞多分化潜能的发现颠覆了人们对其命运的传统认识，然而决定肥大软骨细胞命运的细胞和分子机制一无所知。本项目拟解析肥大型软骨细胞转变过程中的关键细胞生物学过程及ESCRT相关的分子机制。我们首先建立和完善了小鼠肥大软骨细胞及其子代细胞体内示踪技术和流式细胞分选技术，完成了肥大软骨细胞转变为骨髓间质细胞的表型及功能分析。结合10X Genomics单细胞转录组技术和细胞谱系示踪技术，建立了骨骼间质细胞的单细胞转录组，并在此基础上利用新的“Cre-Lox/Dre-Rox”遗传工具小鼠发现了肥大软骨细胞转变过程中关键的骨骼干细胞中间状态。我们还利用多种自噬关键基因的遗传小鼠模型明确了经典自噬通路在肥大软骨细胞转变中的作用，发现了ESCRT依赖的线粒体清除与肥大软骨细胞转变为骨骼干细胞的联系, 为全面理解肥大软骨细胞的生理功能及软骨内成骨的细胞学机制提供了全新的认识。本项目在Bone Research和Cell Research发表标注项目资助的论文2篇。综上所述，本项目完成了研究任务，并取得了显著进展。