近日钟基因核心成分与经典Wnt通路在BMSCs增龄性改变中的交互作用和机制

Aging of bone marrow-derived mesenchymal stem cells（BMSCs）would directly result in the decreased bone mass, thus causing the age-related changes of periodontal and osseous tissues. The previous research clues indicate that the crosstalk of the Canonical Wnt pathway and Bmal1/Per2, the core components among the circadian clock genes, might regulate the aging process of BMSCs. Rev-erbα and Rorα, which are circadian clock genes and meanwhile play critical roles in regulation of cell metabolism, may be the medium of the crosstalk and control the balance and stability between the Canonical Wnt pathway and Bmal1/Per2. To verify the hypothesis, the related clock genes and the Canonical Wnt pathway will be studied during the aging process of BMSCs. Furthermore, the transcription regulation between Bmal1/Per2 and Canonical Wnt pathway will be detected by the Chromatin Immunoprecipitation.And gene transfection and RNAi will be used to change the expression of Bmal1/Per2, Rorα/Rev-erbα, and the activity of the Canonical Wnt pathway. After that, the indices related to osteogenesis, aging of BMSCs, related gene expression profiles and changes of Wnt pathway will be detected. The purpose of this study is to clarify the interaction between Bmal1/Per2 and the Canonical Wnt pathway in regulating the aging of MSCs at cellular and molecular level. Also aimed at supplying experimental foundation for preventing the early aging of periodontal and osseous tissue and seeking new target of treating the senescence-related disease.

BMSCs的增龄性改变，直接导致骨量下降，使骨和牙周组织出现衰老改变。课题组前期研究提示：近日钟基因核心成分Bmal1/Per2和经典Wnt通路的crosstalk可能调控着BMSCs增龄性改变，而Rev-erbα和Rorα可能是该crosstalk的媒介，控制着二者的平衡和稳定。为验证该假说，本研究在检测BMSCs增龄性改变中相关近日钟基因及经典Wnt通路的基础上，使用染色质免疫共沉淀法检测Bmal1/Per2与Wnt/β-catenin通路间的转录调控；并通过基因转染、RNA干扰等分别控制Bmal1/Per2、经典Wnt通路、Rorα/Rev-erbα，检测BMSCs的衰老、成骨及相关基因和信号分子的联动效应，以期从细胞和分子水平阐释Bmal1/Per2和经典Wnt通路在BMSCs衰老改变中的交互作用及其机制，为预防骨及牙周组织早衰提供新思路，也为干细胞的组织工程学和治疗学提供新靶点。

骨衰老的机制在于BMSCs增龄性改变，近日钟基因调控这一过程，但具体作用及机制不明。本研究在检测BMSCs增龄性改变中相关近日钟基因及Wnt/β-catenin通路的基础上，使用ChIP-seq检测Bmal1/Per2的转录调控，并分别控制Bmal1/Per2、Wnt/β-catenin通路、Rorα/Rev-erbα，检测BMSCs的衰老、成骨及相关基因和信号分子的联动效应。结果显示：随着年龄的增长，BMSCs出现衰老表现，增殖及成骨分化能力降低； Bmal1、Per2、Rorα表达降低，Rev-erbα表达增加；Wnt/β-catenin通路活性下降。Bmal1/Per2通过Rorα和Rev-erbα、作用于Wnt/β-catenin通路实现了对BMSCs增龄性改变的控制，其中Bmal1发挥主导作用，但须依赖于Per2。在促增殖、抑衰老方面， Bmal1和Per2有协同作用；在成骨方面，Bmal1有赖于Per2的共同作用，且Bmal1的促进作用较Per2的抑制作用处于更为优势的地位；Bmal1正向、Per2负向调节Wnt/β-catenin，当二者共同作用时，Bmal1占主导地位；Wnt/β-catenin正向调节成骨，正向影响Bmal1、Rorα，负向调节Rev-erbα。当BMSCs被诱导衰老后，Wnt/β-catenin通路激活以代偿，而增强Rorα表达后，激活表现得更明显，以使衰老进程受牵制；增强Rorα的表达，能一定程度逆转衰老，但并不能使细胞完全恢复活力。抑制Rev-erbα表达，能一定程度抑制衰老，提高增殖，Wnt/β-catenin通路的激活可能是其机制。ChIP-seq分析显示，Bmal1及Per2的靶基因与生物过程、分子功能等有关，其靶通路主要与代谢、增殖相关；Bmal1/Per2在Wnt/β-catenin通路的基因上虽有峰的富集，但Wnt/β-catenin可能不是其直接作用的靶通路；Bmal1/Per2对BMSCs成骨/衰老的影响更多是通过mTOR及Hippo通路产生；Bmal1/Per2对相应miRNA也有调控作用。总之，本研究证实Bmal1/Per2-Wnt/β-catenin信号转导途径在BMSCs衰老和成骨过程中起重要作用，并深入探索了其中可能存在的靶向关系，为骨衰老防治提供了新思路。