川芎嗪抑制骨髓间充质干细胞miR-139-5p表达促其向脑缺血损伤区迁移的多靶点作用机制研究

Bone marrow mesenchymal stem cells (BMSCs) have broad application prospects in the treatment of ischemic stroke, and its low mobility and survival rate limit the curative effect. Our previous studies showed that BMSCs pretreated with tetramethylpyrazine (TMP) could increase the mobility and up-regulate the expression of CXCR4, MMP-2, MMP-9, but the mechanism was not clear. It was found that BMSCs pretreated with TMP inhibited the expression of miR-139-5p. The aim of this study was based on identifying the multi-target effects of miR-139-5p, to investigate the effect of TMP on the inhibition of miR-139-5p on BMSCs migration in vitro and in vivo by transfection of BMSCs with miR-139-5p mimic. To clarity the molecular mechanism is related to the inhibition of TMP on miR-139-5p expression and the up-regulated expressions of CXCR4, MMP-2, MMP-9 which are the targets of miR-139-5p. This study is not only helpful to reveal the specific molecular mechanism of TMP in promoting the migration of BMSCs to the brain ischemic injury area based on miRs multi-targeting regulatory molecules, but also to explore the clinical application of stem cells in the treatment of cerebral ischemia.

骨髓间充质干细胞（BMSCs）在缺血性脑卒中治疗中有广阔的应用前景，其低迁移率和存活率限制了疗效。我们前期研究发现川芎嗪（TMP）预处理的BMSCs能提高迁移率，并上调CXCR4、MMP-2、MMP-9的表达，但进一步机制尚不明确。预实验发现TMP预处理的BMSCs能抑制miR-139-5p的表达。本项目拟在明确miR-139-5p多靶点作用的基础上，从体外和体内基于miR-139-5p mimic转染BMSCs，明确川芎嗪抑制miR-139-5p促进BMSCs迁移作用，并进一步阐明分子机制与miR-139-5p靶向CXCR4、MMP-2、MMP-9使川芎嗪抑制miR-139-5p从而上调CXCR4、MMP-2、MMP-9表达密切相关。本项目研究不仅有助于揭示川芎嗪基于miRs多靶向调控相关分子促BMSCs向脑缺血损伤区迁移的具体分子机制，更为探讨脑缺血干细胞治疗的临床应用提供了实验依据。

骨髓间充质干细胞（BMSCs）在缺血性脑卒中治疗中有广阔的应用前景，其低迁移率和低存活率限制了疗效。前期研究发现川芎嗪（TMP）预处理的BMSCs能提高迁移率，并上调CXCR4、MMP-2的表达，但具体机制尚不明确。本研究对川芎嗪预处理和未预处理的BMSCs进行了小RNA测序后，用双萤光素酶报告基因检测miR-139-5p对CXCR4的靶向性；用miR-139-5p mimic/inhibitor或CXCR4过表达质粒转染BMSCs后TMP预处理，划痕和Transwell实验检测细胞迁移能力；RT-qPCR检测miR-139-5p表达量，WB方法检测CXCR4、MMP-2表达变化；采用线栓法诱导大鼠大脑中动脉阻塞模型，尾静脉注射PBS或相应处理（miR-139-5p mimic转染、川芎嗪预处理）后的BMSCs悬液，行为学评分评价神经功能，TTC染色检测脑缺血梗死体积，用免疫荧光法检测迁移到缺血周边区BMSCs数，WB方法检测脑缺血周边区CXCR4的表达。结果显示川芎嗪预处理和未预处理的BMSCs有差异表达的miRNAs；双荧光素酶报告基因检测确认miR-139-5p靶向CXCR4；划痕和Transwell实验结果显示miR-139-5p mimic使BMSCs划痕愈合减慢（P<0.01），迁移细胞数减少（P<0.01），CXCR4（P<0.05）和MMP-2（P<0.05）的表达减少，而miR-139-5p inhibitor的作用正好相反；CXCR4过表达质粒转染BMSCs组促细胞迁移，联合miR-139-5p mimic促迁移作用更强；TMP预处理后BMSCs的miR-139-5p表达量明显减少（P<0.01）；而TMP预处理会逆转miR-139-5p mimic的抑制迁移和使CXCR4和MMP-2蛋白表达减少作用。与TMP+BMSCs组比，TMP+BMSCs+mimic组大鼠脑梗死体积增加（P<0.05），行为学评分升高（P<0.05），向损伤区迁移的数量减少（P<0.01），CXCR4蛋白表达下调。由此阐明川芎嗪调控miR-139-5p靶向CXCR4和MMP-2促BMSCs向脑缺血损伤区迁移。本项目的研究结果明确了川芎嗪促BMSCs向脑缺血损伤区迁移的具体机制与miRNAs相关，且与miRNAs的多靶点靶向作用有关，对指导BMSCs的临床应用具有重要意义。