靶向骨髓间充质干细胞的纳米颗粒递送antago-miR-483-5p治疗骨质疏松的实验研究

Osteoporosis (OP), the growing metabolic skeletal disorder worldwide, has been known as a “silent killer” due to its high rate of incidence and disability in elder population. It is characterized by decreased bone formation and increased bone resorption. Currently, drug selection for osteoporosis is limited. Bisphosphonates, the first-line drugs for osteoporosis due to their low price and high efficiency in reducing the bone resorption, have gastrointestinal side-effects and, more importantly, their long-term use inhibits osteoblast and osteoclast functions. Consequently, new strategies and agents for treatment of OP is urgently needed. Bone marrow mesenchymal stem cells (BMSCs) have the potential to differentiate into various cell types, including adipocytes, chondrocytes and osteoblasts. During osteoporosis progression, osteoblast activity decreases due to reduced differentiation of BMSCs into osteoblasts and increased differentiation into adipocytes. However, the molecular mechanisms behind the shift in BMSC differentiation remain elusive. MicroRNAs (miRNAs) are a class of small (~22 nucleotides), single-stranded noncoding RNAs found in diverse organisms, which down-regulate the expression of target genes by either mRNA degradation or translational inhibition. Recently, several miRNAs were found to be involved in either osteogenesis or adipogenesis. However, most of these miRNAs have only been investigated in vitro, and their functional roles in the pathophysiological mechanisms responsible for bone loss in bone marrow have yet to be established. The roles of miRNAs in the switch between osteoblast and adipocyte differentiation of BMSCs in bone marrow are also unknown. Here, we identified a novel, miRNA, miR-483-5p, that is overexpressed in the BMSCs of ovariectomized (OVX) OP mice but not in control mice. Our preliminary study demonstrates that miR-483-5p regulates BMSCs’ bifurcation into osteoblasts and adipocytes. we propose to use nanoparticles as carriers to deliver BMSC-targeting aptamer-antago-miR-483-5p into OP mice. The nanoparticles will stimulated bone formation in OVX mice by tail vein injection. Furthermore, we will investigate the mechanism of miR-483-5p in regulating BMSCs differentiation. Thus, our study provides a new mechanism and a novel therapeutic target for OP.

骨质疏松（OP）是中老年人群中发病率最高的生长代谢性骨骼疾病，其主要特征为骨形成减少、骨吸收增多。骨髓间充质干细胞（BMSCs）成骨分化减少是骨形成减少的重要原因。我们的前期研究发现在卵巢切除术构建的OP小鼠的BMSCs细胞中miR-483-5p表达水平与骨形成负相关，进一步细胞实验证明miR-483-5p可抑制BMSCs的成骨分化从而抑制骨形成。于是，我们设想体内注射其拮抗剂antago-miR-483-5p可用于治疗OP，但核酸小分子体内应用存在无靶向和易降解等缺点，因此，我们拟制备靶向BMSCs的纳米颗粒递送antago-miR-483-5p，该颗粒经小鼠尾静脉注射即可定向、高效地到达BMSCs细胞并参与成骨调控。在此基础上，本项目拟应用生物信息学和分子生物学等技术，深入阐明miR-483-5p抑制BMSCs成骨分化及骨形成的分子机制，为OP治疗寻找一条新的途径。

背景：促进骨髓间充质干细胞（BMSC）成骨分化是治疗骨质疏松症（OP）的一种有前景的治疗策略。本研究表明 miR-483-5p 抑制 BMSCs 的成骨分化。因此，选择性地将携带antagomir-483-5p（miR-483-5p抑制剂）的纳米颗粒递送至BMSCs有望成为治疗OP的有效手段。.主要研究内容：（a）证明抑制miR-483-5p可促进骨形成：培养小鼠原代BMSCs细胞，real-time PCR 及ELISA等实验证明上调miR-483-5p水平可抑制 BMSCs成骨分化，减少骨形成；抑制miR-483-5p可促进BMSCs成骨分化，增加骨形成。（b） 制备新型包裹agomir-483-5p或antagomir-483-5p的FAM-BMSC-aptamers偶联的纳米颗粒，体外试验检测该纳米颗粒的物理特性及成骨调控功能。（c） 在OVX小鼠中验证上述纳米颗粒调控OP病理进程的作用：构建OVX去势动物模型，尾静脉注射上述制备好的纳米颗粒，通过骨小梁、骨密度等指标验证纳米颗粒对OP病理进程的调控。（d）阐明miR-483-5P抑制骨形成的分子机制：通过miRNAs靶点预测软件Target Scan，荧光素酶分析、siRNA干扰、免疫染色及Rescue等体内外实验验证miR-483-5p对靶基因MAPK1及Smad5表达水平的抑制，最终明确miR-483-5p调控BMSCs成骨分化的分子机制。.重要结果：OVX和老年骨质疏松小鼠BMSC中miR-483-5p水平升高。antagomir-483-5p体外抑制BMSC中miR-483-5p水平可促进ALP、Bglap等骨形成标志物的表达。成功制备携带 antagomir-483-5p 的 FAM-BMSC-aptamer偶联的纳米颗粒，该纳米颗粒可被 BMSCs 吸收，从而在体外刺激 BMSC 成骨分化并在体内延缓OP病理进展。此外，我们的研究表明，MAPK1和Smad5是 miR-483-5p调节 BMSC 成骨分化和OP病理过程的直接靶标。 .科学意义：本研究确立了携带antagomir-483-5p的FAM-BMSC-aptamer偶联的纳米颗粒在OP治疗中的重要作用，这些纳米颗粒可作为临床防治OP的新型候选药物。另外，本研究中优化的小分子核酸靶向给药平台将为临床疾病的治疗提供新思路。