衰老过程中成骨细胞外泌体介导miRNA靶向FOXO1调节血管生成的作用机制研究

Osteoporosis is a serious threat to the health of the elderly population. Decrease in quantity of blood vessel in bone tissue would accelerate osteoporosis. FOXO1 inhibit endothelial cell proliferation through Wnt signal pathway. Previous study had shown that osteoblasts can regulate the surrounding cells, such as endothelial cells, through external secretory pathway. Our pilot experiments found that miRNA-96-5p，-370-5p，-411-5p， -379-5p，-15b-5p were down-regulated in ageing bone and osteoblasts. Prediction analysis showed that these miRNAs might target the gene expression of FOXO1. Thus we proposal here that the exosomes secreted by osteoblasts could ship the above miRNAs into endothelial cells, and then inhibit FOXO1 gene expression and promote angiogenesis, and the down-regulation of the above miRNAs in ageing osteoblast would result in increased activity of FOXO1 and then inhibit angiogenesis, finally accelerate osteoporosis. In this study, we will use aging animal model with exosome inhibitor to explore the effect of exosome secreted by osteoblasts on FOXO1/Wnt signaling and bone angiogenesis in vivo. Moreover, we will use cell co-culture systems to investigate exosome trace and the effect of exosome derived from osteoblasts on the process of angiogenesis; finally, we will combine multiple strategies including exosome tracing, overexpression/inhibition of miRNA, overexpression/inhibition of target gene, to observe the process of miRNA releasing and changes in FOXO1/Wnt signaling pathway. We aim to ultimately clarify the molecular and cellular mechanisms underlying the effect of exosome miRNAs derived from osteoblasts on the process of angiogenesis in aging bone. This project will be greatly helpful for us to understand the pathogenesis of osteoporosis and will provide novel pharmacological and therapeutic targets against osteoporosis.

骨质疏松严重危害老年人群健康，骨组织中血管数量减少可加速骨质疏松进程。FOXO1能下调Wnt信号通路以抑制血管内皮细胞增殖。成骨细胞可经外泌体途径调节其它细胞。前期实验在衰老骨组织和成骨细胞中筛出5个靶向抑制FOXO1且下调的miRNA；由此推测年轻成骨细胞上述miRNA经外泌体途径抑制内皮细胞FOXO1表达，通过激活Wnt通路促进血管生成；衰老成骨细胞这些miRNA下调，致血管生成减少。本项目首先通过建立衰老动物模型，探讨成骨细胞外泌体经FOXO1/Wnt信号通路对骨血管生成的影响；然后用细胞共培养、外泌体示踪等技术在体外观察衰老过程中成骨细胞外泌体对血管生成的影响；最后用miRNA过表达/抑制剂、基因过表达/抑制技术等策略观察成骨细胞兴趣miRNA对内皮细胞FOXO1、Wnt信号通路及血管生成的影响，最终阐明成骨细胞外泌体miRNA干预骨血管生成的具体机制，为防治骨质疏松提供新思路。

骨质疏松的发病机制十分复杂，骨量丢失老年性骨质疏松症的骨组织中不仅有成骨细胞数量的变化，还有血管数量的减少。然后骨微环境中成骨细胞与内皮细胞直接的关系尚未明确，外泌体可以通过携带miRNA调控细胞的衰老、增殖和分化。本研究分析衰老成骨源性外泌体miRNAs调节血管内皮细胞衰老的分子机制。成骨细胞共培养后血管内皮细胞衰老衰老染色的阳性细胞数目较对照组显著增多。共培养48h后Model组的血管内皮细胞中衰老染色阳性细胞数量、凋亡阳性细胞数明显增多，Ki67增殖染色阳性细胞数显著减少；而（Model+GW（外泌体抑制剂））组血管内皮细胞的衰老、凋亡及增殖染色阳性细胞数量与Control组比较无显著变化。通过查阅相关文献及qRT-PCR筛选，找出与衰老成骨细胞共培养后使血管内皮细胞上调表达的兴趣miR-139-5p及miR-214-3p；分离提取成骨细胞培养上清液中的外泌体，通过超微电镜及WB进行鉴定。当加入衰老成骨细胞外泌体后的血管内皮细胞衰老阳性细胞数、凋亡阳性细胞数均显著增多；在衰老成骨细胞分泌的外泌体中miR-139-5p及miR-214-3p的表达较对照组比较明显增多，同时，衰老成骨细胞外泌体干预后的血管内皮细胞中miR-139-5p表达也明显升高。通过miRNA mimic/inhibitor建立内皮细胞中兴趣miRNA高表达或低表达的模型，结果发现转染miR-139-5p 及miR-214-3pmimic后血管内皮细胞的衰老阳性细胞数目、凋亡阳性细胞数目均显著增多、增殖染色细胞数目显著减少、自上室迁入下室的细胞数目明显减少、划痕愈合面积明显减少；转染miR-139-5p及miR-214-3p inhibitor后衰老血管内皮细胞的上述变化得到缓解。最后通过生物信息学软件分析兴趣miRNA的靶基因预测分析结果表明，miR-139-5p可与FOXO1、TBX1等基因的3’-UTR靶向结合。同时，双荧光素酶实验和qRT-PCR结果分析也进一步表证明miR-139-5p与靶基因的相关关系。综上所述，通过成骨细胞与血管内皮细胞共培养体系发现，衰老成骨细胞外泌体miRNA可以通过靶向抑制相关基因的表达，具有加速血管内皮细胞衰老，促进细胞凋亡，抑制细胞增殖、迁移的能力，这为骨质疏松的预防和质量提供了新的思路。