RhoA/ROCK信号参与单轴牵张应力联合纳米纤维排布诱导间充质干细胞向韧带细胞分化的机制研究

One of the main causes of the poor recovery after injury in ligaments is its low level of regeneration. Considering which, a urinary tract cell-scaffold composite with similar ultrastructure to ligaments shall be an efficient and practical solution. The applicant in this research group had successfully designed a biological scaffold with well-defined sets of properties for biomechanical strength and porosity which is remarkably near to the ideal scaffold. The capacity to support cell growth into the scaffold as well as the potential to induce BMSCs’ (bone marrow-derived mesenchymal stem cells) deformation and committed differentiation had been proved previously. Compared to the chemical regulation, the physical stimulation is more controlled and fits better in the in vivo environment. Cytoskeleton remodeling caused by physical stimulation and alignment of nanofiber can regulate tenogenic differentiation of mesenchymal stem cell by mechanotransduction. Since PhoA/ROCK signal pathway is associated with cytoskeleton reconstruction and mechanotransduction, it might be significant in the tenogenic differentiation of mesenchymal stem cell stimulated by pre-stress and stretch. This project is planning to investigate the role uniaxial mechanical stretch and nanofiber matrix alignment on MSC differentiation and cell deformation. By intervention of the key proteins of RhoA/ROCK signal pathway and it’s upstream and downstream paths, further study will be implemented to verify the function and mechanism of the specific pathway in MSC tenogenic differentiation. By retested in vivo, the results and conclusion will improve the understanding and the essential data accumulates in the study of functional tissue-engineered tendon repair.

韧带损伤后功能恢复不良与韧带组织无法有效自我修复相关。组织工程技术可望有效修复，使用具有与韧带组织超微结构相似的生物支架负载骨髓间充质干细胞是一种有效的修复策略。申请人前期制备了有良好力学强度的高孔隙率取向纳米纱支架，与韧带超微结构相似，可促进细胞长入并具有诱导干细胞拉伸、分化的潜力 。与化学刺激不同，物理刺激对干细胞定向分化的作用更仿生更可控。力学刺激和纳米纤维结构引起细胞骨架重构的应力转导作用可能调控干细胞的定向分化。作为应力转导和调节细胞骨架的重要分子，RhoA/ROCK信号应该在取向纳米纤维联合牵张引力诱导间充质干细胞成韧带分化中具有关键作用。本项目拟观察单轴牵张引力联合三维取向纳米纱诱导干细胞形变、分化情况。通过干预RhoA信号及该通路上下游关键蛋白，探寻该信号在干细胞向韧带细胞分化中可能的应力转导机制并结合动物实验予以证明。为韧带损伤的组织工程修复提供必要的理论依据和实验基础。

韧带损伤后功能恢复不良与韧带组织无法有效自我修复相关。使用具有与韧带组织超微结构相似的生物支架负载骨髓间充质干细胞是一种有效的组织工程修复策略。单轴牵张应力通过应力转导机制诱导取向纳米纤维支架表面骨髓间充质干细胞向韧带细胞分化，具体分子途径不清。RhoA/ROCK信号是细胞应力转导的重要分子。通过本项目研究，主要有如下发现：取向纳米纤维支架表面的BMSCs定向排列并拉伸，RhoA/ROCK信号处于预激活状态，具备向韧带细胞分化的潜能。无规纳米支架表面的BMSCs则无此表现。单轴牵张应力作用下，取向纳米纤维支架表面的BMSCs可以向韧带细胞分化，高表达相关基因和蛋白，低表达骨化相关蛋白;在这一过程中，FAK和ROCK持续激活，表达明显上调。单轴牵张应力作用下，无规纳米纤维支架表面的BMSCs可以表达韧带分化相关基因和蛋白，但表达幅度较低（与BMSCs在取向纳米纤维表面静态培养时相似），在这一过程中，FAK和ROCK开始激活，表达上调。该项目证实，纳米纤维排列方式和周期性牵张应力联合作用引起BMSCs内RhoA/ROCK信号的充分活化，可诱导干细胞向韧带细胞定向分化。该项目为韧带损伤的组织工程修复提供必要的理论依据和实验基础。