Notch1信号通路在梅花鹿茸角再生过程中的作用及调节机制

Deer antler is the only mammalian appendage capable of complete regeneration after shedding and an ideal animal model to study the mammalian organ regeneration and wound healing. Notch1 signaling pathway plays an important role in the process of endochondral ossification. Previous studies found that Notch1 was highly expressed in antler mesenchymal cells and chondrocytes, and DAPT could repress the proliferation and differentiation of antler chondrocytes, indicating that Notch1 signaling pathway might be closely associated with the process of antler regeneration. However, the regulation of Notch1 signaling pathway to antler regeneration has not been reported until now. In the project, it plans to examine the expression of Notch1, Rbpj and MAML in sika deer antler and study the effects of Notch1 signaling pathway on the proliferation and differentiation of antler mesenchymal cells and chondrocytes by in situ hybridization, CRISPR/Cas9, flow cytometry, promoter activity assay, chromatin immunoprecipitation, etc. The purpose is to explore the interrelation among Notch1 signaling pathway, ROS, Nox4, YAP and TGFβ1/Smad signaling pathway so as to reveal the function and regulatory mechanism of Notch1 signaling pathway in the process of antler regeneration. These results will provide theoretical basis for further clarifying the molecular mechanism of antler regeneration, increasing yeild of deer antlers, establishing animal models for mammalian organ regeneration and wound healing.

鹿茸是哺乳动物唯一在失去后还能完全再生的附属器官，是研究哺乳动物器官再生的理想动物模型。Notch1信号通路在软骨内成骨过程中起重要作用。前期研究发现，Notch1高表达于梅花鹿鹿茸间充质细胞和软骨细胞中，DAPT可抑制鹿茸软骨细胞的增殖与分化，提示Notch1信号通路可能与鹿茸再生过程密切相关。然而，有关Notch1信号通路对茸角再生的调控目前尚未见报道。本项目通过原位杂交、CRISPR/Cas9、流式细胞术、启动子活性分析、染色质免疫沉淀等方法，研究Notch1、Rbpj和MAML在梅花鹿茸角内的表达及其对鹿茸间充质细胞及软骨细胞增殖与分化的影响，探讨其与ROS、Nox4、YAP和TGFβ1/Smad信号通路间的相互关系，揭示Notch1信号通路在茸角再生过程中的作用及调节机理，为进一步阐明茸角再生的分子机理、鹿茸产量的提高、哺乳动物器官再生及创伤修复动物模型的建立等提供理论依据。

本研究利用原位杂交、流式细胞术、基因过表达、启动子活性分析、RNA-Seq等方法研究Notch1信号通路对鹿茸软骨细胞及间充质细胞增殖与分化的影响，探讨其与Runx2、Nrf2、ROS、钙离子、褪黑素、Activin A、棕榈酸、ZEA、Shh和TGFβ1信号通路间的相互关系。结果发现，Notch1高表达于鹿茸间充质细胞和软骨细胞中。Notch1可促进鹿茸软骨细胞的增殖，加速其向肥大软骨细胞分化，但此效应可被Notch信号通路抑制剂DAPT和Rbpj抑制剂RIN1所阻断。在鹿茸软骨细胞中，Notch1可通过Rbpj转录因子来促进Shh基因的转录，使Smo和Gli1表达增加。抑制Smo或Gli1可减弱Notch1对鹿茸软骨细胞增殖与分化的诱导。Gli1可与Runx2启动子区域结合，介导Notch1信号通路对Runx2表达的调控。添加DAPT或RIN1可减弱褪黑素、TGFβ1和Activin A对鹿茸软骨细胞增殖与分化的诱导，阻止其对Shh信号通路的调控；补充rNotch1可改善棕榈酸对鹿茸软骨细胞增殖与分化的损伤，使鹿茸软骨细胞凋亡率减少。进一步研究发现，抑制Rbpj可使鹿茸软骨细胞中ROS发生堆积，诱导软骨细胞凋亡。同时，抑制Rbpj可因内质网钙释放通道IP3R异常而引起鹿茸软骨细胞中钙离子含量显著增加，诱使细胞中ATP水平及mtDNA拷贝数发生紊乱，降低线粒体膜电位。RNA-Seq分析发现，抑制Rbpj可引起131个基因上调，34个基因下调。在鹿茸间充质细胞中，ZEA可通过减少细胞内GSH含量和增加ROS水平来抑制细胞增殖，延缓细胞增殖周期由G1向S和G2/M期转变，加速细胞凋亡的发生，而激活Nrf2可改善此效应。过表达Rbpj可阻止ZEA对Nrf2表达及转录活性的抑制，且Rbpj可与Nrf2启动子区域相结合。研究结果可为进一步探明梅花鹿茸角再生的调控机理提供依据。