补肾活血方调控间充质干细胞Dnmt3b/Notch信号通路干预骨质疏松性骨折的作用及机制研究

Mesenchymal stem cells (MSCs) are important as initial factors of fracture repair and their normal migration, proliferation and differentiation play an important role in osteoporotic fracture healing. It has been reported that most differentially methylated loci were situated in the genomic regions, which were associated with differentially expressed genes enriched in pathways related to the function of hMSC from patients with fracture, suggesting that DNMTs-driven DNA methylation could be involved in the process of fracture healing. Our preliminary study showed that Dnmt3b was abundantly expressed in callus at the early stage of fracture. In addition, Dnmt3b regulates the function of MSCs via Notch signaling. In this study, the postmenopausal osteoporotic fracture mice models were constructed on the MSCs-specific reporter mice and the MSCs-specific Dnmt3b gene knockout mice. The patterns of the migration, proliferation and differentiation of MSCs via Dnmt3b/Notch signaling pathway during postmenopausal osteoporotic fracture healing were studied in vivo and in vitro. Based on essential pathogenesis of “Deficiency in Origin and Excess in Superficiality” and “Kidney Deficiency and Blood Stagnation” in the early stage of postmenopausal osteoporotic fracture, our group will explore whether Bushen Huoxue Formula could promote the postmenopausal osteoporotic fracture healing via Dnmt3b/Notch signaling pathway in MSCs. This project will further enrich the theory of “kidney governing bone” and bring great theoretical significance and application value to the treatment of postmenopausal osteoporotic fracture with traditional Chinese medicine.

间充质干细胞（MSCs）作为骨折修复的始动因素，其迁移增殖分化对骨质疏松性骨折愈合起着重要作用。研究发现骨折患者MSCs存在差异甲基化位点，且与其功能相关，提示DMNTs驱动的DNA甲基化机制参与骨折愈合。项目组前期发现Dnmt3b在骨折早期骨痂中大量表达，且Dnmt3b可通过调控Notch信号影响MSCs功能。因此，本项目使用MSCs特异性靶标小鼠和MSCs特异性Dnmt3b基因敲除小鼠，构建绝经后骨质疏松性骨折模型，利用体内定向动态追踪技术，研究骨质疏松性骨折愈合过程中MSCs的功能特性，结合体内外研究阐明Dnmt3b/Notch信号通路对其调控作用。此外，基于骨质疏松性骨折初期“本虚标实，肾虚血瘀”的病机，探究补肾活血方是否通过MSCs中Dnmt3b/Notch信号通路促进骨折早期愈合。本课题的实施将进一步丰富“肾主骨”理论，对中医药治疗骨质疏松性骨折具有重大的理论意义和应用价值。

骨质疏松性骨折（OPF）常常引起骨折愈合过程中骨形成迟缓，易发生骨折延迟愈合或骨不连。项目组前期发现Dnmt3b在骨折早期骨痂中大量表达，且Dnmt3b可通过调控Notch信号影响间充质干细胞功能。然而，Dnmt3b在OPF中的表达特征以及对于OPF愈合的影响仍是未知的。此外，补肾活血方（BSHXF）常常用于治疗OPF，且临床疗效确切，然而其作用机制尚不明确。项目组通过（1）构建绝经后OPF模型，通过X-ray、免疫组织化学染色、Real-time PCR以及WB明确骨折愈合进程以及骨折区域Dnmt3b/Notch信号通路、骨痂组织中成软骨基因的表达情况；（2）通过体外分离OPF小鼠骨膜干细胞 (PSCs)进行微球培养，明确OPF小鼠PSCs中Dnmt3b/Notch信号通路以及成软骨基因的表达情况，通过Alcian blue染色以及Col2a1免疫荧光染色明确其成软骨特性，并使用Notch信号通路抑制剂（DAPT）加以验证。（3）使用PSCs特异性Dnmt3b基因敲除小鼠（Dnmt3bGli1小鼠），构建绝经后OPF模型，通过Micro-CT、ABH/OG染色以及骨组织形态计量学，结合Real-time PCR、Western-Blot，明确BSHXF通过Dnmt3b/Notch信号通路调控PSCs成软骨分化，从而促进去骨折愈合。发现（1）OPF组骨折愈合较Control组延迟；骨折区域骨痂中Dnmt3b表达降低，而RBPjκ表达升高；Dnmt3b、成软骨相关基因的mRNA表达水平降低，而Notch下游基因的mRNA表达水平升高。（2）体外PSCs微球培养发现OPF组PSCs中Dnmt3b、成软骨相关基因的mRNA表达水平降低，Notch下游基因的mRNA表达水平升高；Alcian blue染色结果显示OPF组PSCs微球面积较Control组降低，且Col2a1的表达明显减少；DAPT干预后能有效逆转了其成软骨表型。（3）WT+BSHXF组骨折愈合能力较WT+Veh组强，而Dnmt3bGli1+BSHXF组骨折愈合能力较WT+BSHXF组弱。明确（1）Dnmt3b/Notch信号有效调控OPF愈合过程中骨折区域PSCs成软骨分化；（2）补肾活血方通过Dnmt3b/Notch信号通路调控骨折区域PSCs成软骨分化，从而促进OPF愈合。