YAP1在间充质干细胞软骨内成骨中的作用及机制研究

Bone tissue engineering based on endochondral ossification provides a new way to repair large bone defect. However, the success of this approach is hindered by core degradation and an uneven distribution of bone mineral throughout the construct. As an important sensor of mechanical signal, YAP1 (Yes-associated protein 1) transcription activation factor can convert physical characteristics of biomaterials into chemical signals to control stem cell differentiation. Considering the homology of chondroblasts to osteoblasts, lipoblasts, and neuroblasts, we hypothesize that, by studying the function and mechanism of YAP1, it can control the differentiation of MSCs into chondrocytes as well as the following endochondral ossification. To evaluate the effect of YAP1 on endochondral ossification in various stages, MSCs with enhanced or reduced YAP1 function driven by different promoters are developed in vitro, and then the conditional knockout mice are employed to investigate the function of YAP1 in endochondral ossification in vivo. Finally, through scaffold design and gene regulation, the chondrogenic differentiation of MSCs and the following endochondral ossification are observed. We expect to illuminate the mechanism of YAP1’s effect on the chondrocytes differentiation of MSCs in the early stage and the apoptosis of hypertrophic chondrocytes in the late stage. This study will provide us theoretical basis to regulate the bone tissue repair process reasonably, to promote angiogenesis more effectively, and finally to boost new bone formation in the large bone defect.

基于软骨内成骨的骨组织工程为大面积骨缺损的修复提供了新思路，但目前的技术路线存在软骨板退化、骨生成分布不均等问题。YAP1转录共激活因子作为细胞感受机械信号的重要效应因子，可转导生物材料的某些物理性能，如硬度，并影响MSCs向成骨、成脂和成神经细胞方向分化。鉴于成软骨细胞的同源性，我们推测通过对YAP1的作用及机制研究，可以调控MSCs向成软骨细胞方向分化、进而完成软骨内成骨。为验证这一假设，我们首先在体外检验YAP1与软骨内成骨各个阶段的相关性，其次观察YAP1在体内对转基因小鼠软骨内成骨的影响，然后探讨YAP1调控MSCs软骨向分化及肥大软骨细胞凋亡的机制，最后，通过支架设计和基因调控，了解YAP1影响MSCs软骨内成骨的效果。我们期望能够阐明YAP1对MSCs软骨向分化和肥大软骨细胞凋亡的影响及作用机制，从而有目的的调控经由软骨内成骨的组织修复过程，更有效地修复大面积骨缺损。

Hippo信号通路在调控组织发育与细胞增殖分化方面发挥着至关重要的作用，Yes相关蛋白1（YAP1）转录共激活因子是Hippo通路下游核心效应分子，并且能够感受机械应力。本课题利用基因工程、细胞生物学、细胞生物化学、生物信息学等技术，研究YAP1对于某些硬组织来源的细胞，如成软骨、成骨、成牙骨质等相关细胞的分化和矿化的影响，以及对骨代谢相关分子表达的影响，并分别深入探索可能涉及的机制。主要研究内容包括：质粒构建、慢病毒感染得到YAP1不同表达水平的目的细胞，通过成软骨、成骨或炎性因子相关刺激诱导，观察成软骨或成骨以及骨代谢等特异性基因和蛋白表达。进而通过文献调研和生物信息学手段，筛选出转录因子YAP1下游靶基因和调控通路，并进行相关验证。研究结果显示YAP1通过激活经典β-catenin信号通路抑制SOX9表达并负向调控前成软骨细胞软骨向和肥大向分化；接着我们发现YAP1促进了BMP-Smad通路进而促进成牙骨质细胞矿化，并且通过调控BMP通路下游ID1分子的表达来促进成骨细胞分化和矿化；最近我们又发现在炎性条件下YAP1通过抑制NF-KB通路降低了骨吸收介质的表达。这些证据证明YAP1在机体硬组织发育和损伤修复以及骨代谢过程中发挥着重要的作用，并且与细胞内多个重要信号通路间存在紧密相互联系。该研究课题为丰富Hippo-YAP1通路在硬组织发育、骨代谢方面的作用，同时也为YAP1在机体硬组织疾病中的基因调控与基因治疗等相关研究提供了更多的工作基础和理论依据。