可缓释递送miRNA-135基因表达载体的生物活性支架修复眼眶骨缺损研究

The orbital walls are non- weight-bearing bones with limited self-repairing ability under low stress, and thin with poor blood supply, which are not good for bone regeneration. Conventional bio-scaffold could only repair partial orbital bone defect due to poor osteoinductivity. Development of scaffold with high bioactivity has important research significance in orbital bone repair. Previous studies showed that gene bioactive scaffold exhibited significantly osteoinductive activity, and miRNA-135 could regulate stem cells osteogenic differentiation. This project will systematically study the function and regulatory mechanism of bioactive scaffold with sustained release of miRNA-135 expression vector in repairing orbital bone defects. Specifically, the bioactive scaffold with sustained release of miRNA-135 expression vector was prepared. Thereafter, the efficiency of scaffold release and delivery miRNA-135 expression vector will be evaluated, and the mechanism of scaffold regulate stem cells osteogenic differentiation will be explored. Finally, in vivo osteoinductivity and repairing ability of bioactive scaffold will be evaluated by performing rat cranial defects and canine orbital bone defects repairing experiments. This study will systematically explore the function and regulatory mechanism of bioactive scaffold with sustained release of miRNA-135 expression vector in orbital bone defects repairing at the levels of gene, protein as well as small and large animals, and it will develop one new scaffold for orbital bone defects repairing.

眼眶骨为非承重骨，低应力状态下骨自我修复能力有限，眶壁菲薄，局部血供差，不利于骨的再生，常规生物支架不具有良好的骨诱导性，促新骨形成量少，仅部分修复眼眶骨缺损。开发具有高生物活性的支架材料对眼眶骨修复具有重要研究意义。有研究表明基因活性支架可显著增强其骨诱导活性，本课题组证实miRNA-135可调控干细胞骨向分化。本项目将系统性的研究miRNA-135基因活性支架对细胞骨向分化的调控作用机制和对眼眶骨缺损修复的潜能。具体是制备可缓释miRNA-135基因表达载体的生物支架，评价其缓释和递送基因效率，探究其调控干细胞骨向分化的能力及其作用机制，通过大鼠颅骨、犬眼眶壁缺损修复实验评价支架的骨诱导活性和促骨再生能力。从基因-蛋白-小动物-大动物4个层次，多角度探索miRNA-135基因活性支架在眼眶骨缺损修复中的作用机制和修复潜能，为眼眶骨缺损修复开发新的支架材料。

开发基因活性支架是组织再生基因治疗的一个重要环节，基因在体内的释放和转染效率是调控细胞分化的主要因素，前期研究证明miRNA-135可调控干细胞骨向分化，本研究首先制备可高效载带和递送miRNA-135的磁性纳米材料体系，再优选出高效的磁性纳米载体体系制备miRNA-135基因活性支架材料。生物实验研究结果表明N/P比为50时，磁性纳米材料体系载带miR-135 mimics的量为20ug/mg, 该体系转染进入细胞48小时后可显著上调miR-135的表达水平，并且由miR-135激活的下游成骨相关基因（BSP, OCN,OPN）也表现出显著的表达升高。miRNA-135基因活性支架在修复大鼠颅骨标准骨缺损模型3个月后，颅骨缺损处被完整修复，并且缺损组织处并未出现炎症反应。该研究解决了在体内复杂的生物环境下，miRNA-135长期稳定的调控细胞骨向分化的问题，为组织再生基因治疗奠定基础。.此外，本项目的资助下，项目组还优化开发了其它2种支架材料，相关研究成果已整理发表，包括：(1) 具有表面拓扑结构的纳米活性支架制备、成骨效果评估与机制研究；（2）新型可降解聚酯支架材料的开发与成果效能评价。. 从事与本项目相关的已毕业博士2名、硕士1名。已发表标注课题资助号的论文8篇（影响因子大于5分的有4篇），另有2篇投稿中。参加会议交流1次，并做会议发言。