补肾活血汤提取物对MSCs迁移过程中Wnt信号通路的分子机理研究

The Transwell assay will be used to identify the active components of the BuShenHuoXueTang(BSHXT) involved in promoting migration of MSCs. The BSHXT is extracted with petroleum ether,ethyl acetate,absolute alcohol and aqueous respectively.And this active component will be used to research the expression of MSCs related chemotactic factor and its receptor.The influence of Wnt signaling pathway affected by the active component will be analyzed with the RT-PCR,Western Blot etc. Subsequently,the migration capacity of MSCs treated either with Wnt5a over-expression or siRNA against Wnt5a will be analyzed with the luciferase reporter assay and Transwell assay in the further study. We hope this study could further enrich the traditional Chinese theory of "kidney governs bone and marrow giving birth to bone",and offer traditional Chinese medicine the new ideas and theoretical basis in the treatment of refractory bone diseases like bone defect,bone ununion and disuse osteoporosis after long terms immobilization. Meanwhile,the pharmacological activities that BSHXT promotes fracture healing could be found in-depth,and pharmacological evidence in evaluating and screening of chinese medicine in the treatment of bone fractures could be provided.

本项目拟采用Transwell体外迁移实验观察补肾活血汤石油醚、乙酸乙酯、无水乙醇、水溶性4个提取部位对MSCs迁移能力的影响，选取促进MSCs迁移的特定活性部位；根据选取的特定活性部位继续观察补肾活血汤提取物对MSCs相关趋化因子及其受体的表达；采用RT-PCR、Western Blot等方法，研究补肾活血汤在MSCs迁移过程中Wnt信号通路作用的影响；运用荧光素酶报告基因技术，观察Wnt5a基因过表达对MSCs迁移能力的影响；进一步运用siRNA技术，干扰Wnt5a在MSCs的表达，观察对MSCs迁移能力的影响。期望通过本项目的研究，进一步丰富中医"肾主骨"、"髓生骨"理论，从而为中医药治疗骨缺损、骨不连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。同时，深入发现补肾活血汤干预骨折愈合的药理学作用，从而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。

骨缺损、骨不连一直是困扰临床骨科医生的重要问题。在骨折修复过程中，骨髓间充质干细胞（MSCs）向损伤部位的迁移是影响骨折愈合的重要原因。既往研究发现，补肾活血汤对促进骨折愈合有明显的疗效，然而其是否促进了MSCs向损伤区迁移和存活尚不清楚。本项目探索补肾活血汤不同溶剂提取物对MSCs体外迁移的影响，并筛选出补肾活血汤促进MSCs迁移的活性部位，然后从Wnt5a信号通路探讨补肾活血汤促进MSCs迁移的分子机制。随后利用miRNA-seq，从miRNA-mRNA的层面继续深入挖掘MSCs迁移的机理。.结果发现：原代取材培养的第3代MSCs生长良好、形态均一，呈纺锤形或梭形，具有成骨、成脂分化能力，流式显示阳性抗原表达率CD90（99. 84%）、CD44（99. 99%），阴性抗原表达率 CD45（4. 35%），CD43（0.25%）。补肾活血汤采用索氏提取法，得到石油醚、乙酸乙酯、无水乙醇及水提物4 个部位，其中石油醚部位具有明显促进大鼠MSCs体外迁移的作用。Western-blot及细胞免疫荧光结果显示补肾活血汤石油醚部位上调Wnt5a、PKC、PLC、CaMKⅡ，CXCR4、CCR2、IGF2R蛋白的表达。利用慢病毒沉默Wnt5a，发现抑制MSCs迁移，而过表达Wnt5a则促进MSCs迁移。在miRNA-seq及qRT-PCR验证中，发现补肾活血汤能明显抑制miR-539-5p表达。Transwell及划痕实验表明， miR-539-5p模拟物能明显抑制 MSCs迁移。双荧光素酶报告基因实验显示，miR-539-5p能靶向调控Wnt5a的表达。这些结果提示，补肾活血汤促进MSCs迁移，与调节miR-539-5p/Wnt5a信号通路相关。