三维培养微环境调控表观修饰促肌腱干细胞表型维持与再生效用及机制研究

Tendon injuries occur frequently during sports and other rigorous physical activities. Due to their limited vascularity, innervation and cellular content, natural healing of tendon tissue is extremely inefficient. For the lack of knowledge about the regulation mechanism of the tendon differentiation and maturation, tendon stem cells lose the phenotype when cultured in vitro and the regeneration potential for treating the tendon injuries. Therefore, the key point is to find effective methods for in vitro culture of Tendon Stem/Progenitor Cells（TSPCs）. ..Our previous experiments found that TSPCs phenotype and tenogenesis function loss are related to epigenetic regulation. Preliminary experiment indicated that the three-dimensional(3D) culture and differentiation system could restore the phenotype and tenogenesis potential of tendon stem cells in vitro, which is lost when cultured under normal condition. ..The 3D cultured TSPC also improved tendon repair in vivo model. Meanwhile, the 3D cultured environment could affect tenogenesis phenotype associated protein including histone acetylation, DNA, RNA methylation regulation protein expression. Therefore, the purpose of this study is to prove that 3D culture promotes TSPCs stemness and tenogenesis through RNA DE-methylation regulation to maintain the TSPCs phenotype, tenogenesis and promote tendon regeneration, and establish an effective TSPCs culture system in vitro. It is expected that this study will further clarify the molecular mechanism of TSPCs phenotype and tenogenesis maintenance, and provide experimental basis and knowledge for culture methods of tissue-specific stem cells in vitro.

肌腱损伤是常见的运动系统损伤且难以再生。目前肌腱分化成熟的调控机理不明，肌腱干细胞使用间充质干细胞培养体系，体外培养时表型和腱系分化功能丢失，是肌腱组织工程治疗效果有限的原因之一。我们前期实验发现，肌腱干细胞表型和腱系分化功能丢失与表观调控相关，而三维（3D）培养与诱导分化体系可维持肌腱干细胞表型，增强干性及腱系分化的能力，并能逆转与肌腱干细胞表型相关的组蛋白乙酰化，DNA及 RNA甲基化调控蛋白在二维培养时的表达改变。因此，本课题提出假说体内外三维环境通过调控表观修饰，分别维持肌腱干细胞的表型（RNA甲基化）、腱系分化功能及促进肌腱再生（组蛋白乙酰化）。本研究拟结合现有的干细胞生物学、分子生物学和组织工程学手段，建立有效的肌腱干细胞体外培养体系，阐明三维培养微环境对于肌腱干细胞表型维持与腱系分化的表观调控分子机制，并为肌腱组织特异性干细胞的体外培养提供有效培养方案，推动肌腱干细胞的应用。

肌腱损伤是常见的运动系统损伤且难以再生，干细胞与组织工程是具有潜力的治疗策略，但目前效果不佳，目前肌腱干细胞使用MSC培养体系，其表型和腱系分化功能丢失。因此，需要建立有效的肌腱干细胞体外培养体系。本课题在前期研究基础上进一步研究肌腱干细胞体外培养体系的构建，对三维培养微环境肌腱干细胞的调控及其机理进行研究。已发表SCI论文14篇，其中影响因子>5的14篇，第一及通讯作者10篇，申请1项发明专利，已超额完成预期目标。.研究创新点.1) 阐明了肌腱干细胞诱导生物学效应及调控机制，明确了mRNA甲基化及组蛋白乙酰化在肌腱干细胞分化中的关键作用。2) 技术上获得肌腱干细胞体外培养的优化培养条件，构建了肌腱干细胞3D培养体系，验证了其维持肌腱干细胞表型，调控肌腱干细胞分化特征。3) 发现了3D培养微环境通过其表观调控维持肌腱干细胞表型，促进肌腱干细胞分化、成熟的机理。4) 应用获得肌腱干细胞的体外培养体系，维持及诱导肌腱干细胞定向分化，并运用于体内肌腱组织工程，促进肌腱损伤的修复。