川芎嗪预处理促进骨髓间充质干细胞向脑缺血损伤区迁移和存活及机制研究

Bone marrow-derived mesenchymal stem cells（BMSCs）transplantation provides a broad application prospects in the treatment of cerebral ischemia . However, the low rate of migration to ischemic injury sites and survival of BMSCs is one of the major obstacles to limit therapeutic benefit. In recent years，the studies found that the pharmacological pretreatment before transplantation could improve the migration and survival of BMSCs and enhance its therapeutic efficacy. As a traditional and classical meridian-guiding herb which good at helping other herbs to play efficacy in the brain, the active ingredient tetramethylpyrazine pretreatment can promote migration and survival of BMSCs is still not clear. In our preliminary experiments, we found that pretreatment with tetramethylpyrazine could enhance migration and survival of BMSCs in vitro. In this project, we first explore the effects of tetramethylpyrazine pretreated on migration and survival of BMSCs in vitro and in vivo. Then we investigate the mechanisms of migration based on the promigratory factors, including stromal cell-derived factor 1 receptor CXCR4, β1 intergrin VLA-4 and matrix metalloproteinase-2, 9, also the mechanisms of prosurvival based on phosphoinositide-3 kinase(PI3K)/ Akt, extracellular signal-regulated kinases 1/2(ERK1/2) and Bcl-2. Finally, we explore the therapeutic efficacy of intravenous injection of BMSCs which pretreated with tetramethylpyrazine and paracrine mechanisms following cerebral ischemia. These studies provide a new idea for drug selection of pharmacological pretreatment of BMSCs, and offer a new experimental evidence to reveal the scientific essence of helping other herbs to play efficacy in the brain of Chuan Xiong.

骨髓间充质干细胞（BMSCs）移植治疗脑缺血有广阔的应用前景。但是BMSCs向缺血区迁移率和存活率低是限制其疗效的主要原因。近年来研究发现，移植前对BMSCs进行药物预处理能促进其定向迁移和存活，并提高治疗效果。引经药川芎能"引药上行"，其有效成分川芎嗪预处理能否促进BMSCs向脑缺血区迁移和存活尚不清楚。预实验发现，川芎嗪预处理在体外能促进BMSCs迁移和存活。本项目拟在体外和体内，进一步明确川芎嗪预处理对BMSCs定向迁移和存活的影响；然后从基质细胞衍生因子-1受体CXCR4、β1整合素VLA-4和基质金属蛋白酶-2,9探讨促迁移机制，从PI3K/Akt、ERK1/2和Bcl-2探讨促存活机制；最后观察川芎嗪预处理的BMSCs静脉移植对脑缺血的治疗作用，并从旁分泌角度探讨作用机制。本课题研究将为BMSCs预处理的药物选择提供一个新思路，为揭示川芎"引药上行"的科学内涵提供实验依据。

脑缺血是成年人致死致残的主要原因。骨髓间充质干细胞（BMSCs）移植治疗脑缺血有广阔的应用前景。但是BMSCs向缺血区迁移率和存活率低是限制其疗效的主要原因。近年来研究发现，药物预处理能促进BMSCs迁移和存活。引经药川芎能“引药上行”，其有效成分川芎嗪预处理能否促进BMSCs向脑缺血区迁移和存活尚不清楚。本项目首先在体外观察川芎嗪预处理对BMSCs迁移和存活的影响，然后从基质细胞衍生因子-1（SDF-1）受体CXCR4和基质金属蛋白酶-2,9（MMP-2、9）探讨促迁移机制，从PI3K/Akt、ERK1/2和Bcl-2/Bax探讨促存活机制；然后观察大鼠脑缺血后，经尾静脉移植川芎嗪预处理的BMSCs向缺血损伤区迁移和存活情况，以及对脑缺血的治疗作用，并从神经细胞凋亡和血管生成探讨作用机制。结果发现：第3代BMSCs生长良好、形态均一，呈纺锤形或梭形，BMSCs阳性抗原CD29、CD90表达率分别为98.3%、99.5%，阴性抗原CD34、CD45表达率分别为0.1%、1.4%。Transwell小室和划痕实验结果显示，川芎嗪（25、50、100 μmol/L）预处理促进BMSCs迁移和黏附，上调CXCR4、MMP-2、MMP-9 mRNA和蛋白表达，效应可被CXCR4拮抗剂AMD3100或MMPs抑制剂GM6001抑制；在H2O2（500 µM，24 h）诱导BMSCs凋亡的实验中，川芎嗪（25、50、100 μmol/L）预处理24 h，可以增加BMSCs活性，减少活性氧（ROS）产生和细胞凋亡，上调p-AKT、p-ERK1/2、Bcl-2/Bax表达，上述效应可分别被PI3K/Akt、ERK1/2抑制剂wortmannin和PD98059逆转。在脑缺血模型中研究发现，尾静脉移植川芎嗪预处理的BMSCs后，有更多的BMSCs向缺血损伤区迁移和存活，上调SDF-1、CXCR4、VEGF、BDNF、Bcl-2/Bax表达，减少神经细胞凋亡，促进缺血周边区血管新生，同时改善神经功能和减少梗死体积。这些结果提示，川芎嗪预处理促进BMSCs向缺血损伤区迁移和存活，是提高BMSCs治疗潜能的有效策略。