炎症刺激下调控periostin表达的microRNA分子鉴定及其影响成骨细胞分化的机制研究

Nonunion and delayed union of fractures resulted from infection is serious complications in orthopaedics because of its complex condition, long duration, low success rate and high recurrence rate. However, periostin protein is involved in the control of bone formation and bone mineral density. Furthermore, it can regulate the bone strength by influencing the formation of collagen. Our team has demonstrated that decreased expression of periostin when inflammation inhibited differentiation of osteoblasts. Nevertheless, the reversal of osteoblast differentiation activity generated by NBD polypeptide caused up-regulated expression of periostin protein. Similarly, we have found that some microRNA exhibited significant differences under the inflammation stimulation by literature review, which is exactly one of the regulated molecules that may be the modulators in periostin gene expression in our anticipation. Based on inflammation can inhibit differentiation of osteoblasts by down-regulating the expression of periostin, the objective of this project is to find out a new molecular mechanism that targets periostin protein and the reason of the abnormal expression of miRNA. Furthermore, we want to explore the possibility and feasibility of the application of miRNA molecular for reversing the inhibition of inflammation to differentiation of osteoblasts.

感染引起的骨折延迟愈合及不愈合是骨科临床严重的并发症，其病情复杂，病程漫长，治愈率低，复发率高。而Periostin蛋白涉及骨形成的调控以及骨密度，并通过调节胶原的形成参与骨骼强度的调节。本课题组在前期研究中明确了炎症抑制成骨细胞分化时会引起periostin表达下调，而通过NBD多肽逆转成骨细胞的分化活性时会引起periostin蛋白表达的上调。同时查阅文献我们发现某些microRNA表达水平在炎症刺激下存在着显著差异表达，而这些microRNA恰是我们通过生物信息学预测的可能调控periostin基因表达的分子之一。本项目在明确炎症刺激通过下调periostin的表达来抑制成骨细胞分化的基础上深入开展机制研究，拟筛选出可能靶向作用于periostin表达的新分子机制，期望找出miRNA分子异常表达的可能机制，探索应用miRNA分子逆转炎症对成骨细胞分化抑制作用的可能性和可行性。

由感染引起的炎症反应导致骨破坏是骨科临床治疗的难点,其作用机制尚不明确。本课题组在成骨定向分化结合TNF-α处理的细胞模型上和LPS处理小鼠炎症模型上，研究了炎症对成骨定向分化和骨髓间充质干细胞（BMSCs）增殖和成骨定向分化的作用和机制。在细胞水平采用BMP-2诱导C2C12成骨分化细胞模型，研究主要炎症因子TNF-α对成骨定向分化的影响。对细胞转录组数据进行生物信息学分析，发现14个差异miRNA主要参与轴突向导、HIF信号通路、AMPK信号通路等。在LPS诱导小鼠炎症模型上，我们发现LPS所诱发的炎症反应可引起小鼠股骨皮质骨厚度、体积和体积分数显著降低。进一步研究发现，LPS炎症小鼠骨外膜中的成骨标志基因骨钙素（OCN）表达显著下调；组织免疫荧光检测表明，LPS可明显抑制骨外膜细胞中Periostin、p-Akt和非磷酸化β-catenin表达。提示炎症环境下调骨外膜细胞中periostin/Akt/wnt/β-catenin信号，抑制骨外膜干细胞的成骨定向分化，导致皮质骨破坏。此外，LPS还可引起小鼠松质骨量进行性下降，成骨细胞显著减少，干骺端OCN表达下调。提取骨髓细胞进行CFU-F检测，发现LPS对BMSCs并无明显直接效应，而是通过髓系细胞作用间接影响BMSCs增殖。AO/EB染色结果表明，LPS可引起髓系细胞的非凋亡性死亡数量增多，进一步研究发现LPS能显著激活髓系细胞焦亡相关因子IL-1β、IL-18和NLRP3的表达。细胞水平深入研究表明，LPS主要引发巨噬细胞焦亡，释放炎症介质。提示：炎症微环境可能主要通过引发巨噬细胞焦亡释放大量炎症介质，从而抑制BMSCs增殖和更新，导致松质骨量丢失和骨破坏。本课题系列研究初步阐明了炎症性骨丢失的潜在机制，为探索靶向药物调控并改善炎症所致骨破坏具有重要临床意义。