基于miR-17-92/PTEN/PI3K/Akt/mTOR通路研究药对川芎-当归协同 MSCs-exosomes 干预缺血性脑损伤神经再生的效应机制

Neural regeneration after ischemic brain injury is an unsolved problem in the current medical field. Nerve cell proliferation and migration is one key point of neuroregeneration while another one is the axon regeneration. It has a good clinical foundation for the treatment of cerebral ischemia with couplet medicines Chuanxiong-Angelica, and our team have found that this medicine has a positive effect on the proliferation and migration of nerve cells after brain injury. In this study, Chuanxiong-Angelica works synergistically with bone marrow mesenchymal stem cells-derived exosomes to promote axonal regeneration based on miR-17-92 / PTEN / PI3K / Akt / mTOR pathway, which is the key pathway for the regulation of neuronal axon regeneration, in combination with new discovery in the field of stem cells. A series of strategies including a combination of in vitro and in vivo experimental models, such as MSCs-exosomes and neuronal co-culture model, miR-17-92 cluster knockout or over-expression neurons and the MCAO rats model, will be used. Several analytical methodologies will be carried out to explore the intervention effect of couplet medicines Chuanxiong-Angelica and its monomers on the component of MSCs-exosomes as well as synergistic effects and dose-effect relationship of couplet medicines and MSCs-exosomes on promoting neural regeneration. "Component - targets" and molecular mechanism of regulating neural regeneration will be elaborated gradually. Thus, it will provide a possible path and experimental evidence for exploring the cooperative traditional Chinese medicine and stem cells for the treatment of ischemic stroke.

缺血性脑损伤后的神经再生修复是当前医学领域的未解难题，其中两个关键环节是神经细胞增殖迁移，以及神经元的轴突再生。药对川芎-当归治疗缺血性中风临床基础良好，课题组前期发现其对脑缺血损伤大鼠神经细胞增殖和迁移具有促进作用。本项目拟以调控神经元轴突再生的关键通路miR-17-92/PTEN/PI3K/Akt/mTOR通路为切入点，结合干细胞领域新发现，以川芎-当归协同骨髓间充质干细胞外泌体MSCs-exosomes干预轴突再生。以MSCs-exosomes与神经元共培养模型、miR-17-92基因簇过表达和敲减细胞模型、MCAO大鼠模型为观察对象，系统研究川芎-当归及其单体对MSCs-exosomes成份的影响、与MSCs-exosomes协同对神经元轴突再生的干预效应、环节、量效关系，以及调控轴突再生的“组分-靶点”和分子机制；为探索中药协同干细胞治疗缺血性中风提供可能的路径及实验依据。

缺血性脑损伤后神经再生修复是当前医学领域的难点问题。川芎-当归是中医经典药对，二者组成芎归方被广泛应用于临床。干细胞治疗是再生医学中具有广阔前景的新技术。本项目围绕神经再生修复的关键环节----神经轴突再生开展研究，探究川芎-当归协同骨髓间充质干细胞外泌体调控轴突再生的效应和机制，获得以下结论：（1）以芎归方开发的上市中成药舒脑欣滴丸（SNX）与骨髓间充质干细胞外泌体（BMSCs-exosomes）联合干预脑梗死模型大鼠，促进了大鼠神经功能恢复和学习记忆能力；（2）芎归方协同BMSCs-exosomes治疗增加了Nestin+/BrdU+、DCX+/BrdU+、NeuN+/BrdU+细胞数量，促进神经细胞的再生、迁移与分化，增加olig2+/BrdU+新生少突胶质细胞和NF-200蛋白表达，促进了轴突生长；（3）芎归方协同BMSCs-exosomes治疗增加了梗死侧运动皮层M1区和内侧前额叶皮质区椎体细胞突触长度和棘突密度，改善海马区神经元树突棘形态和密度，增加了突触功能相关蛋白SYN和PSD-95的表达，调节了神经突触的可塑性；（4）芎归方筛选的活性成分配比增加了骨髓间充质干细胞培养液中miR-17-92簇的表达，进而调控共培养模型中神经元细胞PTEN/PI3K/AKT/mTOR通路各环节分子的表达，促进神经元轴突生长。本项目的研究成果为芎归方协同干细胞外泌体治疗缺血性中风的临床转化提供了可靠的实验依据；基于经典药对川芎-当归筛选出活性组分配比，并阐明其优化干细胞作用促进神经元轴突生长的机制，为拓宽经典方剂现代开发研究路径作出了有益探索。