miR-582-5p-TGFβ信号调控异常应力下间充质干细胞成骨分化的分子机制及补肾活血方干预作用研究

The change of subchondral bone structure and mechanical properties is the main reason for the formation of osteoarthritis (OA). Repetitive abnormal stress stimulation can induce subchondral bone trabecular microfracture, which will recruit and induce bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation to promote bone remodeling. Eventually, the destruction of bone remodeling balance will lead to bone sclerosis. Many studies have shown that miRNA and TGF-β signaling are involved in the process of osteogenic differentiation of BMSCs. According to the TCM, the basic pathogenesis of OA is the deficiency of kidney and blood stasis. The treatment of OA should use the method of “kidney tonifying and blood circulation”. Stem cells in the human body have the common attribute of the innate essence. Previous study found that the expression of miR-582-5p in subchondral bone of OA was decreased, the loss of TGF-β signaling could induce OA. And Bushenhuoxue recipe could inhibit mouse subchondral bone sclerosis in mouse knee joint to effectively delay the OA process, but the specific regulatory mechanism is not clear. Lentivial vector and exogenous miRNA, siRNA transfection techniques, and real time PCR, Western blot, immunohistochemistry methods are used in this study. The purpose of this project is to study the molecular mechanism of miR-582-5p-TGF-β signaling regulating mesenchymal stem cells osteogenic differentiation under abnomal stress and the possible mechanism of Bushen Chinese medicine on mesenchymal stem cells via the in vivo and in vitro experimental study. This study will provid new research strategies and targets for the prevention and treatment of OA in traditional Chinese medicine.

软骨下骨结构与力学性能改变是骨性关节炎（OA）形成的主要原因。长期异常应力刺激引起软骨下骨微骨折，招募并诱导骨髓间充质干细胞（BMSCs）成骨分化进行骨重塑，最终导致骨硬化。研究表明miRNA与TGF-β信号参与BMSCs成骨迁移分化过程。中医认为肾虚血瘀是OA根本病机，应以“补肾活血”为治则，人体内干细胞具有先天之精属性。前期研究发现miR-582-5p在OA软骨下骨中表达下调，TGF-β信号缺失会诱发OA，自拟补肾活血方能够抑制OA小鼠软骨下骨的硬化，但具体调控机制不明确。本项目拟借助慢病毒载体与外源miRNA、siRNA转染技术，运用Real-time PCR、Western Blot、免疫组化等方法，体内外实验研究miR-582-5p-TGF-β信号调控异常应力下间充质干细胞成骨分化的分子机制，及补肾活血方影响间充质干细胞成骨分化可能作用机理，为中医药防治OA提供新的研究策略和靶点

软骨下骨结构与力学性能改变是骨性关节炎（OA）形成的主要原因。长期异常应力刺激引起软骨下骨微骨折，招募并诱导间充质干细胞成骨分化进行骨重塑，最终导致软骨下骨硬化发生。研究表明miRNA与TGF-β信号参与间充质干细胞成骨分化，项目组研究发现miR-582-5p在OA软骨下骨中表达下调，且miR-582-5p能够通过靶向下游靶基因Runx2来抑制TGF-β信号通路，从而发挥其抑制干细胞成骨分化、促进干细胞成脂分化的功能。中医认为肾虚血瘀是OA根本病机，应以“补肾活血”为治则，人体内干细胞具有先天之精属性。本项目通过体内外研究发现，补肾活血方能够减少OA发病过程中软骨下骨内源性干细胞的分布与数量，抑制软骨下骨及间充质干细胞中miR-582-5p、Runx2、成骨特异性分子ALP以及TGF-β信号关键分子pSmad2的表达，进而延缓OA小鼠软骨下骨硬化的形成。通过本项目的研究，明确了miR-582-5p-TGF-β信号调控异常应力下间充质干细胞成骨分化的分子机制，及补肾活血方影响间充质干细胞成骨分化可能作用机理，为中医药防治OA提供新的研究策略和靶点。