活血Ⅰ号靶向ROCK信号促进骨折后间充质干细胞迁移的分子机制

Inducing the migration of mesenchymal stem cells (MSCs）into fracture area has been a pivotal problem during the speeding up union of fracture. Activating blood circulation to dissipate blood stasis is the therapeutic method in the initial stage of fracture. We have demonstrated Huoxue Ⅰcan induce the migration of MSCs and up-regulate the genetic expression of ROCK2 in our early research. So we set out to explore whether Huoxue Ⅰcan selectively induce the directed migration of MSCs into fracture area through ROCK signaling pathway or not. First, the model in which Huoxue Ⅰinduced the migration of MSCs will be developed. ROCK gene promoter reporter gene will be constructed to explore which component of Huoxue Ⅰactivating ROCK signaling pathway. Furthermore, to investigate the molecular mechanism of ROCK signaling pathway involved in the effect of Huoxue Ⅰon the migration of MSCs, we will employ specific siRNA or inhibitor Y-27632 to block ROCK. Moreover, to further characterize the regulation of chemotactic migration in MSCs by ROCK signaling pathway with HuoxueⅠstimulus, model rats in the initial stage of bone fracture will be established. These techniques of animal living body fluorescence imaging, cell chemotaxis assay, etc. will be used. Studying ROCK signaling pathway may help us elucidate the functionary mechanism about activating blood circulation to dissipate blood stasis in the fractured therapy from the view of the migration of stem cells, and provide a new strategy to the therapeutic method in the initial stage of bone fracture syndrome.

如何促进间充质干细胞（mesenchymal stem cells, MSCs）向骨折端迁移是加快骨折愈合的关键问题。活血化瘀为中医骨折早期治法，前期工作发现，活血Ⅰ号可促进MSCs迁移，上调ROCK2基因表达。因此，提出“活血Ⅰ号有效成分群通过选择性调节ROCK信号通路促进MSCs向骨折端定向迁移”。①以活血Ⅰ号促进MSCs迁移为模型，构建ROCK基因启动子报告基因，确定活血Ⅰ号中何种成分激活ROCK信号通路；②运用特异性siRNA或抑制剂Y-27632阻断ROCK信号通路，阐明ROCK信号通路介导活血Ⅰ号促进MSCs迁移的分子机制；③运用动物活体荧光成像、细胞趋化等技术观察活血Ⅰ号对骨折早期模型大鼠MSCs迁移作用及ROCK信号机制。从干细胞迁移ROCK信号通路角度阐明中医活血化瘀法在骨折治疗中的作用机制，为中医活血化瘀法在骨折早期治疗中的应用提供新思路。

如何促进间充质干细胞向骨折端迁移是加快骨折愈合的关键问题。活血化瘀为中医骨折早期治法，前期工作发现，活血Ⅰ号可促进MSCs迁移，上调ROCK2基因表达。因此，提出“活血Ⅰ号有效成分群通过选择性调节ROCK信号通路促进MSCs向骨折端定向迁移”。本研究结果发现：（1）活血Ⅰ号（红花挥发油）促进MSCs迁移机制与ROCK信号通路相关，通过上调ROCK2、MLC基因表达，增加细胞收缩功能。（2）活血Ⅰ号（红花挥发油）不仅促进MSCs增殖、体外成脂，还可以诱导大鼠体内异位成脂分化。活血Ⅰ号还可促进小鼠皮肤损伤修复。（3）活血Ⅰ号的有效成分硬酯酸甲脂、棕榈酸、棕榈酸甲酯、α-亚油酸均对大鼠BMSCs具有明显的迁移作用。棕榈酸处理大鼠BMSCs LncRNA sequence结果显示，与MSC迁移相关基因RP11-324O2.3、RP11-505K9.1、AC072062.3上调。棕榈酸促进hMSCs迁移的机制可能与LncRNA Wnt-5a-AS有关。（4）活血Ⅰ号的单体之一，硬脂酸甲酯（SAME）通过VAV1/ROCK2通路促进BMSCs迁移；动物实验表明：SAME 通过动员内源性MSCs至外周血，迁移至损伤部位，从而促进软骨损伤修复。共发表学术论文4篇，其中包括3篇SCI，培养两名硕士研究生，撰写2篇毕业论文。已申请两项国家发明专利：一种红花挥发油的应用；一种硬脂酸甲酯的应用。本项目从间充质干细胞迁移角度出发研究，初步揭示活血Ⅰ号促进骨折后间充质干细胞迁移的分子机制，为骨折三期辨治原则提供物质基础。活血Ⅰ号是一个潜在的促进间充质干细胞向脂肪分化的有效中药复合物，为临床软组织的修复与再生、丰乳、美容等带来新希望，可用于以非治疗目的的面部年轻化、隆胸、隆鼻和隆臀术中，及修复或矫正软组织损伤。通过硬脂酸甲酯促进了间充质干细胞的迁移和促进软骨损伤修复，提供一种中药活性小分子单体在制备治疗软骨损伤药物中的应用。