miR-124通过Notch/BMP通路调控骨髓间充质干细胞向神经细胞分化的机制研究

This research is aimed to explore molecular mechanism of bone marrow mesenchymal stem cells (BM-MSCs) regulated by miRNA,and repairing efficacy of regulated BM-MSCs for spinal cord injury. Previously, authors had made sure key microRNAs such as miR-124 which regulated neural differentiation of BM-MSCs with methods of miRNA chips, realtime fluorescent quantitation PCR and bioinformatics softwares. Furthermore, preliminary experiment showed over-expression of mi-R124 could raise expression of NSE and MAP-2 in differentiated BM-MSCs. Software forecasting showed Notch, HES1 and BMP which related with signal pathway of Notch /BMP, were target genes of mi-R124. Based on previous research, we plan to construct expression plasmid of miR-124 and anti-miR-124 which transfect BM-MSCs, then luciferase report system was used to confirm target genes , and RT-PCR and western blot to detect expression of target genes. For the second step, BM-MSCs with chemical modification of miRNA ASO or viral vector transfection of miRNA will be transplanted into rats with spinal cord injury to confirm their function of repairing neural injury. This research conduce to clarify molecular mechanism of miR-124 regulating neural differentiation of BM-MSCs, provide theoretical and experimental proof for inducing neural differentiation of BM-MSCs.

本研究旨在探讨miRNA参与调控骨髓间充质干细胞（BM-MSCs）神经分化的分子机制及经miRNA调控的BM-MSCs修复脊髓损伤的效果。申请者前期初步筛选出miR-124可能是BM-MSCs向神经分化的关键miRNA。同时发现BM-MSCs向神经分化过程中miR-124表达升高，且过表达miR-124上调神经元标志物NSE，MAP-2的表达。进一步软件预测发现miR-124靶向Notch /BMP信号通路的关键蛋白Notch、HES1、BMP。本项目将通过荧光素酶法、western bloting及蛋白芯片验证靶基因，同时检测靶基因相关通路上关键分子的表达变化。最后用大鼠脊髓损伤模型，观察经miR-124调控的BM-MSCs对神经损伤的修复作用。本研究将阐明miR-124通过Notch /BMP通路调控BM-MSCs神经分化的机制，为miRNA水平诱导BM-MSCs神经分化提供实验依据。

脊髓损伤的修复是长期困扰医学界的科学研究难题, 迄今没有公认有效的治疗方法，细胞移植促进脊髓功能恢复，成为最具前景的脊髓再生手段。BM-MSCs具有跨胚层分化功能，在一定条件下可分化为神经细胞，因此移植BM-MSCs治疗SCI已经成为目前脊髓再生研究领域的热点。申请者利用miRNA芯片法、RT-PCR结合生物信息学分析技术确定BM-MSCs向神经分化的关键miRNA：miR-124；通过构建miR-124慢病毒载体系统，上调BM-MSCs的miR-124表达；miR-124过表达促进BM-MSCs向神经样细胞分化，分化的细胞突起变得细长，呈现典型的双极、三极或者多极细胞形态；免疫荧光和RT-PCR检测发现分化的细胞神经元标志物NSE，MAP-2表达显著增强；荧光素酶实验显示miR-124能与靶基因notch1的3’UTR结合，抑制靶基因的表达，提示notch1是miR-124的靶基因；Western blot检测发现mir-124可以下调notch1的表达，抑制抗神经分化因子hes1和hes5的表达，抑制神经分化抑制因子NeuroD 的表达，提示miR-124通过下调Notch信号通路促进BM-MSCs向神经细胞分化的机制；BM-MSCs和miR-124调控的BM-MSCs均增加大鼠脊髓损伤模型损伤处神经元细胞数量，神经轴突数量，促进损伤脊髓功能修复，并且miR-124调控的BM-MSCs组BBB评分显著优于BM-MSCs组。本研究提供了一种BM-MSCs向神经细胞分化的新方法，为miRNA水平诱导BM-MSCs神经分化提供实验依据。本研究获得的miR-124调控BM-MSCs具备取材方便、易于体外扩增、不易引起免疫排斥反应及伦理学争议、能修复损伤神经组织等特点，有可能成为修复脊髓损伤的理想种子细胞。