miR-21调控脑缺血神经前体细胞存活和神经再生分子机理研究

Neural stem cells are promising therapy for stroke patients. The promotion of endogenous neurogenesis contributes to the behavioral recovery after stroke. However, most newly generated neuroblasts die in the ischemic boundary zone, which minimizes its contribution to functional recovery after stroke. In our preliminary data, we for the first time discovered that the expression of miR-21 was significantly increased in ischemic SVZ neural progenitor cells (NPCs). In addition, we also found miR-21 inhibited the apoptosis of NPCs, indicating miR-21 could regulate the cell survival and neurogenesis in SVZ NPCs after stroke. To verify our hypothesis, a middle cerebral artery occlusion (MCAo) model was employed. By means of laser capture microdissection, miRNA in situ hybridization, double fluorescent immunostaining, we will determine the expression of miR-21 in NPCs after stroke. Second, by means of luciferase reporter, electroporation, we will examine whether miR-21 regulates apoptosis signaling pathway through its target genes. Finally, using a novel miR-21 knockout transgenic mouse and lentivirus vector, we will further in vivo verify the effect of miR-21 on the survival, neurogenesis and neurological functional recovery after stroke. We believe our study will make a unique contribution to molecular mechanism underlying stroke-induced neurogeneis as well as provide novel targets and avenues for the treatment of stroke.

神经干细胞是治疗脑缺血非常有希望的方法，促进内源性神经干细胞再生有助于神经功能的恢复，但是由于新生的神经细胞多在缺血区死亡，对神经功能的恢复有限。在前期研究中，我们首次发现小分子miR-21在脑缺血神经前体细胞（NPCs）中表达明显增高，预实验显示miR-21能够抑制NPCs的凋亡，因此我们推测miR-21可能参与了调控缺血后神经干细胞存活和神经再生。为证实这一假说，我们将建立动物大脑中动脉梗塞模型，应用激光显微切割、miRNA原位杂交、荧光双染色等技术观察miR-21在NPCs中的表达；应用荧光素酶报告基因、核转染技术等探讨是否miR-21通过抑制靶标基因调控凋亡信号转导途径；最后应用小分子RNA基因敲除动物和慢病毒载体，在体观察miR-21基因对脑缺血后新生神经细胞存活、神经再生和神经功能的影响。本研究将会为脑缺血神经再生研究提供新的理论基础，并为治疗脑缺血提出新的靶点和途径。

神经干细胞是治疗脑缺血的新方法，促进内源性神经再生有助于脑缺血后神经功能的恢复，但是由于新生的神经细胞多在脑缺血区死亡，对神经功能的恢复有限，因此揭示神经再生的分子调控机制十分重要。应用动物大脑中动脉脑梗塞模型、转基因动物、激光显微切割、miRNA原位杂交、核转染、实时荧光定量PCR、慢病毒基因载体、荧光双染色等技术， 本课题首次发现和揭示：1）小分子RNA-21(miR-21)在脑缺血恢复期神经前体细胞中表达明显增高；2）miR-21在神经前体细胞中表达的时空演变；3）miR-21能够抑制神经前体细胞的凋亡和促进神经干细胞存活；4）miR-21通过抑制靶标凋亡基因Programmed cell death protein 4 （PDCD4）和FASL表达，促进存活基因Bcl-2升高调控凋亡信号转导途径；5）首次在国内外建立了小分子RNA-21特异性神经前体细胞基因敲除动物； 6）研究证实了miR-21可以成为防治脑缺血促进神经功能恢复的新靶点，7）提供了基于RNA分子治疗脑缺血的新途径。本研究将会为脑缺血神经再生研究提供新的理论基础，并为治疗脑缺血提出新的靶点和途径。