川芎嗪刺激骨髓间充质干细胞分泌高表达miRNA-486-5p外泌体促进脑缺血后神经血管再生及机制研究

The replacement of stem cells by exosomes is the hot topic in recent years. Exosomes mainly regulates the function of target cells through transferring miRNA, but the exosomal miRNA is selective packaging and is affected by extracellular stimulation. Tetramethylpyrazine (TMP) is a pharmacologically active component from the meridian-guiding herb Rhizoma Chuanxiong. Our previous studies have found that exosomes with high expression of miRNA-486-5p derived from tetramethylpyrazine-stimulated bone marrow mesenchymal stem cells enhance neurovascular regeneration after ischemic stroke, but the mechanism is not clear. In this project, by using methods of pharmacology, molecular biology and bioinformatics in vivo and in vitro, we firstly further confirm that exosomes derived from TMP-preconditioned BMSCs improve neurovascular regeneration after cerebral ischemia. Then we verify that exosomes derived from TMP-preconditioned BMSCs exert its effect through transferring miRNA-486-5p. Finally, we prove that the effect exosomes derived from TMP-preconditioned BMSCs is mediated by miRNA-486-5p direct targeting PTEN and its signal pathway PI3K/Akt. This project not only reveals the molecular mechanism of TMP-preconditioned BMSCs promoting neurovascular regeneration after cerebral ischemia, but also provides a new strategy for stem cells therapy.

外泌体替代干细胞治疗是近年来研究的热点。外泌体主要通过运输miRNA调控靶细胞功能，但外泌体内miRNA是选择性包装，并受到细胞外刺激的影响。我们前期研究发现，引经药川芎的有效成分川芎嗪（TMP）预处理骨髓间充质干细胞（BMSCs），分泌高表达miRNA-486-5p的外泌体，促进脑缺血后神经发生和血管生成，但机制尚不清楚。本项目拟在体内和体外，采用药理学、分子生物学和生物信息学等方法，进一步确认TMP预处理的BMSCs外泌体促进脑缺血后神经血管再生；然后明确TMP预处理通过上调BMSCs外泌体miRNA-486-5p表达发挥效应；最后证明miRNA-486-5p通过调控靶基因PTEN及信号通路PI3K/Akt介导TMP预处理的BMSCs外泌体效应。本项目研究不仅揭示了TMP预处理的BMSCs外泌体促进脑缺血后神经血管再生的分子机制，还为干细胞治疗提供一个新策略。

脑缺血是成年人致死致残的主要原因。干细胞移植是治疗脑缺血的重要策略。与干细胞相比，外泌体具有安全性好、易通过血脑屏障、易贮存运输和无伦理限制等优点，有望替代干细胞治疗。前期研究发现，川芎嗪（TMP）预处理的骨髓间充质干细胞（BMSCs）促进脑缺血后神经血管再生，并分泌富含miR-486的外泌体，但其是否通过分泌富含miR-486的外泌体发挥作用尚不清楚。本项目首先确认BMSC-Exos是否通过转运miR-486促进脑缺血后神经血管再生，然后明确TMP预处理是否刺激BMSCs分泌富含miR-486的外泌体促进脑缺血后神经血管再生。结果发现：（1）BMSC-Exos促进脑缺血后神经发生和血管生成；（2）BMSCs转染miR-486 mimic/inhibitor，脑微血管内皮细胞和神经干细胞分别转染PTEN siRNA，证实BMSC-Exos通过转运miR-486靶向抑制PTEN促进神经发生和血管生成；（3）与BMSC-Exos比较，TMP-BMSC-Exos显著改善神经功能，抑制神经元凋亡，促进神经发生和血管生成，上调miR-486、Bcl-2/Bax、p-Akt、VEGF和BDNF表达，抑制PTEN表达，miR-486 inhibitor能抑制上述效应，而PTEN抑制剂bpV则逆转miR-486 inhibitor效应。（4）与BMSC-Exos比较，TMP-BMSC-Exos增强OGD/R损伤的脑微血管内皮细胞增殖、迁移和管形成，上调miR-486、p-Akt、Ang-1/Tie-2、VEGF/VEGFR2蛋白表达，下调PTEN表达，miR-486 inhibitor可抑制上述效应，而PTEN siRNA则逆转miR-486 inhibitor效应。结果提示，TMP预处理刺激BMSCs分泌富含miR-486的外泌体靶向抑制PTEN，促进脑缺血后神经发生和血管生成，为脑缺血的治疗提供一个新策略。