MicroRNA-21在大鼠急性脊髓损伤与修复中的作用及相关机制研究

The spinal trauma is always accompanied with complete or incomplete spinal cord injury, in which the secondary injury is aggravated by posttraumatic circulatory disturbance and cell apoptosis. MiR-21 is an important regulatory gene for cell apoptotic inhibition and angiogenesis process. Our preliminary study have indicated that miR-21 was differentially up-regulated after spinal cord injury, which contributed to the protection of cord parenchyma and neural fuction recovery. However, miR-21, as an upstream controlling gene, is needed to further investigated on its distinctive functioning mode and molecular mechanism. In this study, we will plan to recombinate the miR-21 lentivirus for the treatment of injured spinal cord. In vitro, a model of oxygen-glucose deprivation (OGD) will be established within the Mouse Umbilical Vein Endothelial Cells (MUVECs) line and neuron cell line.The biological behaviour of the MUVECs and neurons will be examined after the recombinational lentivirus transfection. In vivo, based on the rat spinal cord injury model, the lentivirus will be locally injected into the injured spinal cord to up-regulate or down-regulate the miR-21 expression.The parameters of the angiogenesis and cell apoptosis will be examined, as well as the downstream target gene expression. Based on the two level of study in vivo and vitro, the whole experiment aims to figure out the positive effects and reveal the relationship between the miR-21 treatment and the neural repairing, proceeding to expound the upstream miR-21 regulatory mechanism in the SCI process. All the above-mentioned investigations are expected to establish the theoretical basis and experimental evidences of the pre-clinical value of the miR-21 as a therpeutic agent in the mannagement of SCI.

严重的脊柱创伤常伴有完全或不完全的脊髓损伤（SCI），创伤后微循环障碍和神经细胞凋亡是加重SCI的两大重要因素。改善SCI后血供、抑制神经细胞的凋亡是促进脊髓损伤后修复的重要手段。miR-21是抑制细胞凋亡、促进血管生成的重要调控基因，我们的前期研究证实miR-21在大鼠急性SCI后的表达上调，并有效促进SCI后神经功能的恢复，但其具体调控方式和作用机制不明。本项目拟通过构建慢病毒载体，分别转染体外内皮细胞及神经元细胞糖氧剥夺模型，同时体内局部转导大鼠损伤段脊髓，通过上调或下调内源性miR-21的表达,观察其对SCI后血管生成和神经细胞凋亡以及相关下游靶基因表达的影响。通过体内、体外两个层面的研究，明确miR-21在急性SCI过程中的调控作用，为阐明miR-21在SCI后血管生成和神经细胞凋亡中的生物学机制提供新线索，进而为miR-21治疗急性SCI的临床前评估提供理论依据和实验基础。

急性脊髓损伤（ SCI）是一类严重的运动系统创伤，改善 SCI 后血供、抑制神经细胞的凋亡是促进SCI修复的重要手段。本课题系列研究了miR-21在改善脊髓损伤后血管网络再生，减少神经元凋亡，促进轴突再生，改善神经功能恢复中的作用及机制。主要研究内容及结果：（1）发展了脊髓微血管及神经元的3D可视化与量化分析，揭示不同节段脊髓血管网络的形态特征与差异；发现脊髓前角神经元分布密度显著高于后角。（2）miR-21在SCI后血管内皮细胞和神经元中表达持续升高；miR-21在胚胎期脊髓组织中表达显著高于出生后，揭示miR-21在大鼠SCI后表达的时空分布及脊髓胚胎发育中表达变化。（3）miR-21可显著改善SCI后运动功能评分及运动诱发电位，提高对机械、热与冷刺激的感知，证明了miR-21对SCI后神经功能恢复的保护作用。（4）细胞实验证实miR-21可促进运动和感觉神经元的轴突再生， 动物实验证实在SCI后不同时间点，miR-21可促进损伤区域的轴突再生和皮质脊髓束再生，明确了PTEN在其中发挥重要作用，揭示了miR-21促进SCI后轴突再生的作用及机制。（5）细胞实验证实miR-21可促进OGD模型中内皮细胞的存活、迁移与成管，抑制内皮细胞的凋亡，在动物实验中，miR-21可促进SCI后血管的新生，增加SCI后微血管分支节点密度、血管容量和微血管节段密度，揭示TIMP3在其中发挥关键作用，揭示miR-21促进SCI后血管新生的作用及机制。（6）细胞实验证实miR-21可抑制OGD模型中PC-12细胞的凋亡，在动物实验中，发现miR-21可抑制SCI后神经元细胞的凋亡，揭示PDCD4在其中发挥关键作用，揭示miR-21抑制SCI后神经元凋亡的作用及机制。综上，本研究首次系统阐述了miR-21能有效促进SCI后血管新生、微血管3D构象重塑及神经轴突的再生，抑制神经元凋亡，进而改善SCI运动与感觉功能，揭示TIMP3/MMP、PTEN和PDCD4等靶基因在其中发挥关键作用。本研究为miR-21应用于SCI及相关脊髓疾病治疗的临床前疗效评估提供了实验科学依据。在该课题经费支持下共培养研究生9名，中青年骨干医师4名，衍生出国家基金委资助课题2项。发表相关中、英文论文21篇，其中SCI收录19篇（ 总IF=61.053）。参与相关的国内、外重要学术会议10余次，学术获奖2次。