压应力加载介孔支架中LncRNA-NEAT1调控ADSCs成骨分化的机制研究

Bone defect is one of the serious impair of bone surgery. In recent years, tissue engineered bone compose of adipose-derived stem cells (ADSCs) and the novel mesoporous materials provides a new idea for the repair of bone defects. Research shows that the mechanics of microenvironment is an important factor in the regulation of osteogenic differentiation of ADSCs. However, it is still unclear about the regulation mechanism of this course. Our previous studies found that cyclic compressive stress can enhance the osteogenic differentiation of ADSCs in the novel mesoporous material of m-MPC. Gene chip screening found that compressive stress stimulation resulted in changes of gene expression profile in ADSCs. Inhibition of lncRNA-NEAT1 led to osteogenic differentiation of ADSCs significantly. Preliminary results suggest that the cyclic compressive stress may regulate osteogenic differentiation of ADSCs by NEAT1，which further adjustes its downstream target miRNA and osteogenic related signal pathways. In order to reveal the underlying mechanism, following studies intend to clarify the regulating role of NEAT1 and its target miRNA and signal pathway, reveal the mechanism of the stress environment of mesoporous material on osteogenic differentiation of ADSCs, explore the new method of promoting bone defect repairing through stress loading or key factors of signal pathway.

骨缺损是骨外科面临的严重危害之一。近年来脂肪间充质干细胞 (ADSCs)与介孔材料复合形成组织工程化骨为骨缺损修复提供了全新思路。研究表明：力学微环境是调控ADSCs在支架上成骨分化的重要因素。课题组前期合成了一种新型介孔材料m-MPC，研究发现：周期性压应力可增强m-MPC上ADSCs的成骨分化。基因芯片筛选发现：压应力刺激导致ADSCs内lncRNA表达谱发生改变。抑制lncRNA-NEAT1表达促进ADSCs的成骨分化。提示压应力可能通过NEAT1调控下游miRNA和成骨信号通路，继而影响ADSCs的成骨分化。为了进一步验证此假设，本研究拟通过压应力加载ADSCs/m-MPC三维培养模型，明确NEAT1对下游靶标miRNA及成骨信号通路的调控作用，阐明介孔应力环境促进ADSCs成骨分化的机制，探索通过调控应力加载及信号通路中的关键因子，促进骨缺损修复的新方法。

脂肪干细胞与新型介孔支架结合在骨组织工程中的应用为治疗大段骨缺损提供了新的思路和视角，很大程度上克服了传统骨移植的缺点与不足。本研究制备了PCL-PEG-PCL复合材料和带有比表面积为428.0m2和孔隙体积为0.40 cm3/g的m-MS，通过溶剂浇铸-粒子沥滤法合成了 m-MS/PCL-PEG-PCL(m-MPC)复合物；同时通过酶解法从SD大鼠白色脂肪组织分离出脂肪干细胞（ADSCs），将细胞接种于m-MPC上。结果表明，随着m-MPC中m-MS含量的增加，含40w%m-MS的复合材料抗压强度(48MPa)和弹性模量(548MPa)最高；此外由于加入了m-MS，m-MPC的亲水性、降解率和体外生物活性也随之增加，表现出良好细胞相容性。具有生物活性的m-MPC在生物医学领域显示出作为新一代促进骨再生生物材料的广阔前景。.静水压力(HP)是人体骨细胞受到的的一种恒定应力，构成了四分之一的系统血压并且动态调节骨组织的稳态。本研究构建了静水压力下培养的ADSCs/m-MPC 三维支架体外模型，通过基因芯片筛选发现，HP能抑制lncRNA-NEAT1同时增加lncRNA-PAGBC表达来促进ADSCs的成骨分化。进一步研究发现lncRNA-PAGBC作为竞争性内源RNA(ceRNA)，能与成骨抑制性 miR-133b结合，并通过沉默miR-133b增加RUNX2的表达，从而促进ADSCs成骨分化。本研究结果最终表明：静水压刺激可通过lncRNA-NEAT1和PAGBC调节ADSCs的成骨分化，二者作用相反；静水压刺激通过PAGBC/miR-133b/RUNX2轴促进脂肪间充质干细胞成骨分化，其中静水压通过升高lncRNA-PAGBC的表达，从而抑制miR-133b对RUNX2的抑制作用，促进RUNX2的转录翻译，最终表现为促进脂肪间充质干细胞成骨分化。研究压应力作用下 ADSCs 成骨分化过程中信号转导和基因调控的确切机制，对于探索骨缺损修复及骨折愈合的最佳应力环境、应力在骨折愈合与重建过程中发挥作用的机制、高性能组织工程骨的构建、以及介孔材料的临床应用研究有巨大的社会经济价值。