包裹miRNA-320的PLGA壳聚糖复合纳米微球对股骨头坏死的疗效及其ceRNA机制的实验研究

The femoral head necrosis is intended to have higher risk of progression and collapse. The key point of early intervention is to take effective measures to improve the bone regeneration of femoral head before the collapse of the femoral head, and promote the repair of necrotic bone. MicroRNA (miR) acts as a regulatory nonencoding RNA, with stable expression in vitro. With the implication of Low expression of miR-320 in the necrosis of the femoral head and its effect on the regulation of bone regeneration and lipid metabolism. This study intends to investigate the osteogenesis and adipogenic effect of miR-320 to bone marrow mesenchymal stem cell (BMSC) and the underlying change of circle RNA of hsa-circRNA4193, hsa-circRNA6449, hsa-circRNA8796, hsa-circRNA14718. The objective is to establish a miR-320 based network of competing endogenous RNA (ceRNA) for the change of BMSC. Furthermore, micro PLGA/chitosan microspheres coated with miR-320 was prepared to verify their in vitro release profiles and release kinetics. In vitro study, the effect of the composite microspheres on the differentiation of BMSC cells into osteogenesis differentiation will be studied, and the related genomic changes after miR-320 microspheres regulation is also studied. Furthermore, we use the PLGA/chitosan composite microspheres coated miR-320 to the animal model of femoral head osteonecrosis to observe the local osteogenesis regeneration and necrotic tissue repair by histological and radiological study. The importance of this study may provide a new idea for early treatment of femoral head necrosis, and lay a theoretical and technical foundation for the gene therapy for femoral head necrosis.

股骨头坏死的早期治疗重点是促进坏死骨修复。基于我们前期研究，发现在股骨头坏死骨组织中miR-320显著低表达。miR-320可能通过竞争性内源RNA（ceRNA）网络调控影响BMSC参与股骨头坏死发生。.本研究拟验证miR-320家族成员诱导BMSC成骨分化、成脂分化的能力，并在细胞水平验证miR-320家族成员的表达与4个目标环状RNA：hsa-circRNA4193、hsa-circRNA6449、hsa-circRNA8796和hsa-circRNA14718之间的相互关系。构建miR-320对BMSC分化影响的竞争性内源RNA网络。进一步采用聚乳酸-羟基乙酸共聚物(PLGA)/壳聚糖的复合微球作为载体，包裹miR-320，通过体外试验及动物试验观察复合纳米微球对BMSC成骨能力分化的影响及对股骨头坏死的治疗作用。本研究的为股骨头坏死的早期治疗提供新的思路。

股骨头坏死好发于中青年人群，病因包括大量使用糖皮质激素、长期酗酒、创伤等。一旦发生激素性股骨头坏死，2年内的塌陷率为78.6%，近年来随着系统性红斑狼疮患者使用激素治疗比例的增高，激素性股骨头坏死引起了人们的重视。越来越多的研究发现，髓芯减压联合骨髓间充质干细胞（hBMSCs）移植对早期股骨头坏死有一定的治疗效果，但是患者BMSCs 受损的增殖和成骨分化能力影响了早期治疗效果。目前BMSCs细胞功能变化的机制不完全清楚，因而缺乏有效的、针对性的早期治疗措施。近年来，非编码RNA在BMSCs细胞增殖和分化中的作用日益明了并愈受重视。本研究拟通过转录组测序对比分析，运用生物信息学方法分析激素性股骨头坏死患者和对照组患者（发育性股骨头发育不良）BMSCs间差异表达的miRNA和circRNA，进一步通过免疫荧光、qPCR、染色、WB等方法观察其相互调控及其在BMSCs成骨、成脂分化中的作用，进一步研发可长期缓释ncRNA的水凝胶材料，分别在细胞和动物水平观察缓释ncRNA水凝胶材料的安全性和骨修复性能。