可降解二维黑磷介导microRNA调控人脂肪干细胞成骨分化及其机制研究

The key to bone defect regeneration is to actively mediate osteogenic differentiation of stem cells and finally achieve new bone formation. microRNA plays an important role in regulating bone formation and osteogenic differentiation of stem cells. However, naked microRNA is easily degraded by RNA enzyme in the physiological environment, and it is difficult to penetrate the cell membrane into the cells. Therefore, it is necessary to build nano-carriers with high efficiency and low toxicity to protect and deliver microRNAs. This project attempted to use black phosphorus (BP) to construct a biodegradable microRNA delivery system. We will systematic study the synthetic methods of BP nanosheets and quantum dots and study the effect of particle size and surface modification of BP on its physical and chemical properties and biological effects. Then we will study the effect of particle size of black phosphorus on the efficiency of cellular uptake ability and the target gene silencing efficiency of microRNA. We will also investigate the effect of microRNA delivered by black phosphorus on the osteogenesis of stem cells and new bone formation in vitro and in vivo. The successful implementation of this project is to build a BP based microRNA delivery system with high efficiency and good biocompatibility, and to realize the effective meditation of osteogenesis of adipose stem cells and new bone formation in vivo and in vitro. This paper provides new ideas and theoretical basis for the construction of a new generation of nucleic acid carrier which is suitable for tissue repair.

介导干细胞成骨分化形成新骨是实现骨再生修复的关键。micorRNA（miRNA）对骨形成及干细胞成骨分化具有重要的调控作用，然而裸miRNA存在稳定性差、难以穿透细胞膜等问题，限制了其深入研究和应用。本项目拟基于新型二维纳米材料黑磷，发展一种生物可降解的miRNA 输送体系，并开展其调控脂肪干细胞成骨分化行为及机制研究。项目拟研究黑磷纳米片及量子点的制备及修饰方法，并探讨尺寸和表面修饰对其理化性能及生物学效应的影响；构建黑磷输送miRNA体系，并研究其与miRNA的结合机理、跨膜转运和细胞内运输及降解代谢的机制；在细胞和动物层面系统研究黑磷介导miRNA对人脂肪干细胞成骨能力的影响并探讨其作用机制。本项目的成功实施，可构建一种高效低毒miRNA输送体系，实现黑磷介导miRNA对脂肪干细胞成骨分化以及骨缺损再生修复的有效调控，为新一代适用于组织修复的核酸载体的构建提供新思路和理论依据。

由于创伤、骨病或骨肿瘤引起的骨缺损是影响人类生命和健康的重大医学难题。虽然传统的骨缺损修复材料可以部分满足替代缺损部位的需求，但难以满足血管长入及功能化等需求，仍然无法实现骨组织在结构和功能上的完全修复，即再生重建。诱导干细胞成骨分化生成新骨是实现真正意义上“再生”的重要标志。因此，研制可调控干细胞行为和骨组织再生过程的生物活性材料是再生修复的重点。miRNA在调控干细胞分化、骨形成及其内稳态等生命活动中发挥着重要的作用。且相比于生长因子，miRNA结构简单、易于合成及操作、不易失活、且能特异性地从基因水平调控细胞的行为，使其成为近十年来医药领域最热门的研究方向之一。miRNA能否高效发挥效用极大程度依赖于纳米输送载体的选择和设计。因此，本项目旨在构建一种基于二维黑磷的可降解miRNA输送载体，并探究其调控干细胞成骨分化及作用机制。本项目成功制备了表面由磷酸钙矿化层修饰的黑磷纳米片及氨基钛配体修饰的黑磷纳米片和黑磷量子点；通过探讨黑磷基材料在细胞和机体内的降解代谢行为，揭示了黑磷基材料独特的生物降解活性；通过研究黑磷基材料对干细胞及其它多种细胞系增殖、黏附等行为的影响及作用机制，发现了基于黑磷降解活性的独特生物学效应；证明黑磷基载体可高效运载miRNA进入干细胞，并发挥基因调控功能，并在体内外探讨了miRNA对干细胞成骨分化及骨形成的影响及作用机制。本项目的研究结果可为miRNA在骨修复领域的研究应用及纳米运输载体的设计制备提供理论依据和新思路。