肌肉来源细胞构建组织工程化肌腱的关键因素及相关分子机制

Capability of potent proliferation and tissue formation is the key to choose proper seed cell source for engineering functional tendon tissue. Skeletal muscle and tendon are associated during early stage development, they subsequently form different types of tissues under the influnce of different environmental factors. Our pilot study demonstrated that muscle derived cells (MDCs) could form much stronger and more mature engineered tendons than tenocytes (TCs) in the subcutaneous environment (hypoxia relative to skeletal muscle). Based on tendon developmental origins and our pilot study findings, we hypothesize that MDCs can differentiate into TCs and form functional tendon tissue when they are provided with essential tendon development-related environmental factors. This study will employ in vitro cell culture, in vitro and in vivo tendon engineering models to investigate effects of hypoxia, unilateral mechanical stretch, and GDF-8 on the switch of MDCs to TCs and on the formation of stronger engineered tendon as well as the underlying molecular mechanisms. Additionally, we will also utilize techniques of GDF-8 knock-down and overexpression, p38MAPK signaling interference, and in vivo mechanical stretch mediated tissue remodeling to further explore how these factors affect functional tendon formation, collagen superstructure formation, tendon mechanical property and the expression of tendon related extracellular matrix molecules. The success of these studies will set a solid scientific base for the use of MDCs to build sturdy functional tendons in potential clincial applications.

良好扩增和组织形成能力是选择构建功能性肌腱种子细胞的重要因素。骨骼肌和肌腱发育同源并在不同环境因素作用下形成各自的组织类型。预实验发现肌肉来源细胞（MDCs）在皮下环境(相对低氧)中能比肌腱细胞(TCs)形成结构更成熟、抗张力更强的肌腱组织。根据发育生物学原理和预初实验结果，我们提出科学假说：模拟肌腱发育微环境因素能将MDCs转化为TCs并形成功能性肌腱组织。为此，本研究将采用体外细胞培养体系和体内外肌腱构建模型分别探索低氧、力学刺激和GDF-8等肌腱微环境重要因素对MDCs向TCs转化及形成相对高力学强度肌腱的作用与相关机制，并采用包括GDF-8 knock-down和过表达，p38MAPK信号介导和体内力学重塑等手段探索上述因素对功能性肌腱形成、胶原超微结构形成、力学性能和肌腱相关细胞外基质表达等的作用。从而为MDCs这种新型种子细胞构建高力学强度肌腱及临床应用奠定坚实的科学基础。

种子细胞是功能性肌腱构建的核心要素，不仅要易于获取、具有良好的扩增能力，而且还应具备良好的组织形成能力。前期研究中我们发现与肌腱发育同源的肌肉来源细胞（MDCs）不仅可再生出肌腱样组织，而且形成的工程化肌腱力学强度较高。其形成高力学强度肌腱组织的机制可能是MDCs在低氧环境及力学刺激下分泌了适于肌腱形成的相关因子，但缺乏直接支持证据。并且，MDCs从获取方式上比皮肤成纤维细胞复杂；不具备干细胞的多项分化的潜能，那么MDCs与现有细胞相比是否具有明显优势呢？目前尚不清楚。因此，本课题首先将MDCs与常见的种子细胞进行了综合比较，全方位证明了MDCs的优势；同时进一步用差速贴壁技术、基因芯片技术等方式证明了低氧及力学刺激对MDCs形成工程化肌腱的关键作用，并发现了对工程化肌腱形成可能有关的基因。本课题研究结果将为组织工程肌腱的临床转化及进一步优化其构建环境及力学强度奠定了基础。