微纳结构通过FAK/YAP/自噬通路调控BMSCs成骨向分化的分子机制研究

Micro-nano structured biomaterials can cause autophagy, which is worth in-depth study for its role in osteo-differentiation and cellular homeostasis. Our previous study (NSFC81500894) showed the focal adhesion kinase-FAK and Hippo signaling pathway participated the regulation of cell osteo-differentiation caused by nanotopography: FAK-Y397 phosphorylation was strikingly inhibited on nanotopographical surfaces while YAP shifted from cell nucleus to cytoplasmic. FAK and Hippo-YAP both are engaged in autophagy, in order to clarify the regulatory mechanism of FAK/YAP/autophagy signaling in cell osteogenic differentiation on micro/nano-structured surfaces, we aim to detect the alteration of FAK expression and phosphorylation, as well as the key members of autophagy pathway by implantation models in rats and transfected cell model. Meanwhile, the drug loaded implants, gene-silencing technology and chemical blocking/agonist are used to investigate the autophagy related effect of nano-topography on FAK and Hippo pathway, which reveals the role of autophagy in cell osteodifferentiation on nano-surface, and furtherly indicates a novel molecular mechanism of the correlation among FAK, Hippo pathway and autophagy. The project provides theoretical background for design and fabrication of the biomaterial surface which is appropriate for bone repair and of great significance.

生物材料表面微纳结构可引发干细胞自噬(Autophagy)，且影响细胞成骨向分化的作用机制值得深入研究。本项目前期研究（NSFC81500894）表明，黏着斑激酶（FAK）及Hippo-YAP参与调控微纳结构刺激干细胞成骨向分化过程，FAK及FAK-Y397磷酸化均被显著抑制且YAP发生核内外迁移；进一步研究及文献证实FAK与Hippo-YAP均参与自噬的发生，且成骨向分化过程中自噬上调，但其相互之间的关系尚不清晰。本项目拟在此基础上计划采用体内种植体植入、病毒稳定转染细胞、基因沉默和化学阻断/激动剂，结合基因和蛋白表达检测，研究微纳结构激活自噬通路及调节FAK和Hippo-YAP的作用，重点寻求自噬通路过程中的机理，进而获得三者之间相关性的全新分子机制。项目源于多学科交叉的共性问题，立足机体对材料应答的主要科学方向，将尝试为探索适宜骨修复的生物材料表面结构设计提供新的理论参考。

生物材料表面微纳结构可引发细胞自噬(Altophagy)，且影响细胞成骨向分化，而自噬过程如何参与细胞成骨分化的调控尚不清楚。在本项目资助下，我们构建了光滑、微米和纳米级粗糙度三种不同的钛表面拓扑结构，发现纳米拓扑结构表面细胞具有最高的自噬水平，β-catenin核定位最强，而YAP表达水平较低。进一步的研究发现纳米拓扑结构通过自噬介导YAP蛋白降解，促进了β-catenin核转位增加从而激活细胞成骨分化。阻断自噬导致YAP上调，限制了β-catenin核转位。最后，我们对纳米拓扑结构引发细胞自噬的机制做了初步探究，发现纳米拓扑结构通过下调FAK水平从而促进细胞自噬发生。本项目立足于机体对材料应答的主要科学方向，初步阐明了自噬途径在纳米拓扑结构调控细胞成骨分化中的分子机制，为探索适宜骨修复的生物材料表面结构设计提供新的理论参考。