Hedgehog信号通路对梅花鹿茸角再生的调控

Deer antlers are the only mammalian appendages capable of repeated rounds of regeneration and the ideal animal model to study mammalian organ regeneration and wound healing. Hedgehog signaling pathway plays an important regulatory role in the process of chondrocyte proliferation and differentiation. Previous studies found that IHH, Ptc and Smo were highly expressed in the mesenchymal cells and chondrocytes of deer antler, indicating that Hedgehog signaling pathway might be closely associated with the differentiation of antler mesenchymal cells and chondrocytes. However, the regulation mechanism of Hedgehog signaling pathway to deer antler regeneration has not been reported until now. In this project, sika deer antler is used as a model to examine the expression of HH, Ptc, Smo and Gli in deer antlers and the effects of Hedgehog signaling pathway on the proliferation and differentiation of mesenchymal cells and chondrocytes of deer antler by in situ hybridization, immunohistochemistry, gene overexpression, RNA interference, etc. It aims to explore the relationships between these molecules and deer antler regeneration-associated genes (Runx2, TGFβ2, Zfp521, etc.) so as to reveal the function and mechanism of Hedgehog signaling pathway in the process of deer antler regeneration. These results will provide theoretical basis for further clarifying the molecular mechanism of deer antler regeneration, increasing yeild of deer antlers, establishing animal models for mammalian organ regeneration and wound healing.

鹿茸角是哺乳动物唯一在失去后还能完全再生的器官，是研究哺乳动物器官再生及创伤修复的理想动物模型。Hedgehog信号通路在细胞增殖与分化过程中起重要作用。前期研究发现，IHH、Ptc和Smo高表达于鹿茸间充质细胞和软骨细胞中，提示Hedgehog信号通路可能与鹿茸间充质细胞和软骨细胞分化密切相关，然而，有关Hedgehog信号通路对茸角再生调控的机制目前尚未见报道。本课题以梅花鹿为研究对象，通过原位杂交、免疫组织化学、基因过表达、RNA干扰等方法对HH、Ptc、Smo和Gli在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进行研究，探讨这些分子与茸角再生相关基因Runx2、TGFβ2、Zfp521等的关系，揭示Hedgehog信号通路在茸角再生过程中的作用及调控机理，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、RNA干扰、流式细胞术等方法，研究Hedgehog信号通路分子在梅花鹿茸角中的表达及其对鹿茸软骨细胞增殖与分化的影响，探讨其与Activin A、Foxa及TGFβ1信号通路间的相互关系。结果发现，Dhh高表达于鹿茸软骨细胞中。添加rDhh可促进鹿茸软骨细胞的增殖，诱使S期细胞增多。沉默Ptch1可解除其对Smo的抑制，导致Smo激活。补充Smo抑制剂Cyclopamine可消除Dhh对软骨细胞增殖的诱导。Dhh通过Smo来诱导鹿茸软骨细胞向肥大软骨软骨分化，使Gli1-3的表达增加。抑制Gli可减弱Dhh对软骨细胞分化的影响，使Foxa1-3的表达减少。转染Foxa siRNA后，软骨细胞分化标志分子Col X和Runx2在rDhh处理的软骨细胞中的表达显著减少。Shh与受体Ptch1结合后，可解除其对Smo的抑制，活化的Smo进一步激活Gli，通过调控Foxa的转录来诱导鹿茸软骨细胞的增殖与分化。Activin A可激活Shh信号通路，抑制Shh信号通路可减弱rActA对鹿茸软骨细胞的促增殖效应，使软骨细胞向肥大软骨细胞分化受阻。TGFβ1与受体结合后可通过Smad3来增强Shh、Ptch1、Smo和Gli的转录。转染Shh和Gli siRNA或添加Smo抑制剂Cyclopamine及Gli1抑制剂GANT58后，TGFβ1对鹿茸软骨细胞增殖与分化的影响明显减弱；而补充rShh可改善因TGFβ受体和Smad3抑制所导致的鹿茸软骨细胞增殖与分化的障碍。抑制Shh信号通路可减弱Activin A和TGFβ1对Foxa基因的调控。添加rIhh后，鹿茸软骨细胞的增殖活性显著增强，S期细胞增多，但软骨细胞的分化却明显减弱。过表达Ihh可使鹿茸软骨细胞向肥大软骨细胞分化受阻，而沉默Ihh却可促进鹿茸软骨细胞的分化。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。