肌腱细胞外基质和拓扑结构诱导人脂肪干细胞成肌腱分化的实验研究

Human adipose tissue derived mesenchymal stem cells (hAT-MSC) is rich in their resource, and great potential of multiple differentiation (including tenogenic differentiation) and proliferation. We propose the hypothesis: Extracellular matrix (ECM) produced by cultured tenocytes is able to induce tenogenic differentiation of hAT-MSCs and Biglycan and Fibromodulin are likely to be the key inductive molecules. Combination of key inductive molecules and microgrooved topography is able to better mimic the physical and chemical induction signals of the tendon niche environment and thus to promote tenogenic differentiation of hAT-MSCs in a 2D culture model. Further, biomimic tenogenically inductive scaffold developed based on this concept combines with mechanical loading signaling can further optimize the signal mimicking of tendon in vivo niche environment and thus promote tenogenic differentiation and tendon formation by hAT-MSCs in a in vivo setting. We plan to isolate and enrich hAT-MSC using CD45-/CD34+/ CD31- as the markers, and also use decellulized ECM produced by cultured tenocytes to serve as an inductive coating. Additionally, coating with recombinant tendon ECM molecule and the siRNA gene silence of ECM molecules will also be conducted to narrow down the key inductive ECM molecules. Moreover, combination of ECM induction with physical inductive signals of topographic structure and mechanical loading will be our ultimate strategy for comprehensive mimicking of tendon niche environment to prove our proposed hypothesis.

人脂肪间充质干细胞（hAT-MSC)来源丰富、扩增能力强和具有向肌腱分化的能力。我们提出科学假说：腱腱细胞产生的胞外基质（ECM)能够有效诱导hAT-MSC向肌腱细胞分化，而Biglycan/Fibromodulin是关键的诱导分子。关键ECM分子与刻纹表面结构相结合能更加有效综合模拟肌腱微环境中的理化诱导信号，促进hAT-MSC在二维模型中成肌腱定向诱导分化。利用该理念制备三维诱导支架复合力学刺激信号能精确综合模拟肌腱组织所处的体内微环境从而促进ASC在三维模型中的肌腱形成。我们拟采用CD45-/CD34+/ CD31-作为标志物和间充质细胞培养体系分选、富集和扩增出hAT-MSC作为研究对象，采用肌腱细胞产生的胞外基质脱细胞后作为EMC铺层，以及重组ECM分子铺层和siRNA沉默ECM关键分子基因等手段锁定关键的诱导分子及探讨其相关机制，并与拓扑、力学物理信号相结合验证此综合模拟假说。

完成了hAT-MSCs的获取，根据本课题组的前期工作基础和经验获取了多个样本来源的人脂肪间充质干细胞（hAT-MSC)。但因在磁珠富集CD34+， CD45-细胞的过程中细胞数量损失较大，细胞活性降低，不利于后期的实验进展，后续我们直接采用人脂肪干细胞进行实验。在二维水平证实了肌腱细胞体外分泌基质和肌腱脱细胞凝胶对 hAT-MSCs 成肌腱诱导分化的作用，肌腱胞外基质对AT-MSC成脂、成骨和成软骨分化的影响各异。肌腱胞外基质有利于AT-MSC成肌腱分化和成骨分化，不利于成脂分化，对成软骨分化无明显影响。同时发现真皮基质的作用与肌腱脱细胞基质的作用类似，但其诱导成肌腱的正向作用不及肌腱脱细胞基质。在此基础上完成了肌腱细胞外基质成肌腱诱导成分的鉴定，我们尝试用各种肌腱胞外基质中常见的成分单独或两两组合在培养皿上铺层，检测肌腱细胞标志物的基因表达如前。结果显示单一分子铺层时COL1和Fmod铺层诱导效果较好，二者混合铺层时诱导效果有明显的协同作用，但无论采用何种基质铺层，I型胶原的表达都没有显著的上调。因此后续我们采用COL1+Fmod混合铺层。进一步地我们完成了二维模型上综合模拟肌腱细胞微环境胞外基质和拓扑结构信号，在微米级水平材料和纳米级水平的材料上都成功实现了诱导AT-MSC体外成肌腱分化。.在三维水平我们探索了模拟肌腱细胞微环境胞外拓扑结构信号促进hAT-MSC成肌腱诱导分化，同时完成了体内三维水平使用平行纤维纳米材料或在材料上修饰细胞外基质成分透明质酸修复大鼠或兔跟腱缺损的实验研究。