补肾活血法促进骨折修复与重建过程中MSC迁移能力的分子机理研究

The fluorescence imaging will be used to detect the migration of exogenous bone marrow mesenchymal stem cells(BMSCs) induced by BuShengHuoXueFa(BSHXF) to the fracture site during the bone fracture repairment and restitution in the item. Microsceope and immuno-histochemical method, cell immunofluorescence, molecular biology, and serum pharmacology method will be used to understand the molecular mechanism, and fracture local microenvironment will be improved by BSHXF at different time after fracture. It could encourage the secretion of chemotactic factor and the expression of correlated receptor, and regulate the cellular signal transduction,for example, PI3K,PLC,MAPK signaling pathway, and so on, then induce the migration of MSCs to the fracture site. The study will offer the therapeutic target for drug screening at shortening fracture healing time, decreasing fracure complication, preventing and curing bone defect, fracture delayed union or bone nonunion, osteonecrosis and disuse osteonecrosis after fracure prolonged immobilization. MSCs from the bone marrow will be selected to explore the contribution of BSHXF on the migration, proliferation and differentiation potency to bone formation during the bone fracture repairment and restitution. It will affluence the traditional Chinese medicine theory of the kidney being in charge of bone and the marrow giving birth to bone.The contribution of BSHXF on MSCs migration, proliferation and differentiation potency during the prophase, metaphase and anaphase of fracture will be studied, it will offer the theoretical evidence for three phases determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs of traditional Chinese medicine fracture.

本项目运用荧光成像系统检测补肾活血法促进骨折修复与重建过程中外源性骨髓MSCs向骨折端迁移情况。采用显微镜联合免疫组化、细胞免疫荧光、分子生物学、血清药理学技术，了解补肾活血法改善骨折后不同时相骨折局部微环境的分子机制，包括促进趋化因子分泌及相关受体的表达，并通过调节PI3K、PLC、MAPK等细胞信号转导通路，进而促进MSCs向骨折部位定向迁移，可望为缩短骨折愈合时间、减少骨折并发症，防治骨缺损、骨折延迟愈合或骨不连、骨坏死及骨折长期制动后出现的废用性骨质疏松等难治性骨病的药物筛选提供治疗靶点。选择骨髓源性MSCs，探讨补肾活血法促进骨折修复与重建过程中MSCs迁移、增殖、成骨分化能力的影响，丰富了中医 "肾主骨"、"髓生骨"理论。通过研究补肾活血法对骨折早期、中期、后期MSCs迁移、增殖、分化的影响，为"中医骨折三期治疗"原则提供理论依据。

本项目在中医“肾主骨”、“髓生骨”理论指导下，选择骨髓源性MSCs，探讨补肾活血法促进骨折修复与重建过程中MSCs迁移、增殖、分化能力的影响，揭示中医治法方药改善骨折后局部微环境的分子机制及与时间相关性，从而为“中医骨折三期治疗” 原则指导下的中药复方治疗骨缺损、骨折延迟愈合或骨不连、骨坏死及骨折长期制动后出现的废用性骨质疏松等难治性骨病提供新思路和理论依据。主要研究内容为：补肾活血汤及其拆方（含药血清）对骨髓间充质干细胞（BMSCs）增殖及成骨分化的影响、补肾活血汤对不同时相外源性BMSCs迁移能力的影响、补肾活血汤对骨折微环境中趋化因子（MCP-1、SCF、SDF-1、IGF-2）及BMSCs表面相关受体（CCR2、c-kit、CXCR4、IGF-2R）表达的影响、补肾活血汤对PI3K/PLC/MAPK等信号途径的影响及补肾活血汤含药血清对外源性BMSCs迁移能力的影响等。研究发现：补肾活血汤促进骨髓间充质干细胞增殖的有效部位在补肾组乙酸乙酯部位，且以100 μg/mL为最佳药物浓度；补肾活血汤全方含药血清有促进大鼠骨髓间充质干细胞增殖的作用，其中体积分数为10%含药血清的作用最显著；补肾活血汤可以促进大鼠骨折愈合过程中BMSCs的体外迁移，其相关机制可能与上调CCR2的表达，激活MCP-1/CCR2信号轴有关；补肾活血汤促进BMSCs 体外迁移的有效部位为石油醚部位，其相关机制可能与上调CXCR4 蛋白表达，激活SDF-1/CXCR4 信号轴有关；补肾活血汤含药血清可促进BMSCs的体外迁移，并影响CXCR4蛋白的表达，其相关机制也可能与上调CXCR4的表达，激活SDF-1/CXCR4信号轴有关。通过本项目的研究，初步发现补肾活血治法方药干预骨折愈合的部分药理学作用靶点，为今后中医临床治疗骨折药物的评价与筛选提供药理学依据。