突触核蛋白（α-SN）在脊髓可塑性中的作用及其miRNA调控机制研究

SCI (Spinal cord injury) often leads to serious neurological dysfunction and high mutilation. As a common disease in clinical, neurosurgery and orthopedic. It needs developing to clarify the mechanism for the research of SCI based on it’s significant scientific value and practical importance. It is known that several factors following injury occurred and take part in repair after SCI, but the mechanism for the repairing awaits to be elucidated also. Some documents have indicated that spinal cord possess the capability for neural plasticity, and the role of alpha synapse nucleoprotein (α-SN) and micRNA-138. Based on the findings of α-SN and micRNA-138 in the spinal cord following SCI by proteomics research and micRNA chip analysis ,we established the model of transection spinal cord in rat, together with the technologies of gene cloning, RNA interference, immunocytochemistry, RT - PCR, Western Blot, nerve tracking to detect the effect of alpha SN and miRNA-138 on neuroplasticity following SCI .There could be involved in neuronal apoptosis, axon regeneration, synaptic plasticity and rat hind limb movement change. Moreover, we would like to explore whether or not micRNA-138 can regulate α-SN directly. The possible results would be useful to know the role of micRNA-138 in α-SN, in neuroplasticity, so as to provide experimental basis for research regulation mechanism of SCI. The results of this research are also benefit for us to find new strategy in promoting SCI function repair, with important scientific significance and potential value.

脊髓损伤（SCI）是临床神经外科常见的疾病，严重影响神经功能，致残率极高。开展SCI机制研究有重要的科学价值和现实意义。SCI后存在损伤和z自身修复的双重调节因素，而修复机制研究是寻找促进功能重建策略的重点和难点。我们前面的研究显示，SCI后脊髓局部存在一定的可塑性能力，但其是否受α突触核蛋白（α-SN）的影响及miRNA-138调控仍不清楚。本项目在课题组前期蛋白组学及micRNA芯片发现SCI大鼠脊髓内α-SN与micRNA-138变化的基础上，拟建立大鼠脊髓横断伤模型，采用基因克隆、RNA干扰、免疫细胞化学、RT-PCR、Western Blot、神经束路追踪及电镜等技术，研究α-SN及miRNA-138在SCI大鼠脊髓神经元凋亡、轴突再生、突触可塑性和大鼠后肢运动变化中的作用，并探讨miRNA-138是否对对α-SN进行调控，为SCI修复治疗及调控机制研究提供理论依据。

脊髓损伤（spinal cord injury, SCI）是临床神经外科常见疾病，严重影响患者神经功能。修复机制研究是寻找促进功能重建策略的重点和难点。我们前面的研究显示，SCI后脊髓局部存在一定的可塑性能力，但是否受α突触核蛋白（α-SN）的影响及miRNA-138调控仍不清楚。本项目在前期蛋白组学及micRNA芯片发现SCI大鼠脊髓内α-SN与micRNA-138变化的基础上，通过细胞培养和建立大鼠脊髓横断伤模型，采用基因克隆、RNA干扰、免疫细胞化学、RT-PCR、Western Blot、神经束路追踪及行为学等技术，从体内外展开研究α-SN及miRNA-138在SCI大鼠脊髓神经元凋亡、轴突再生、突触可塑性和大鼠后肢运动变化中的作用，并探讨miRNA-138是否对α-SN进行调控。体外研究表明，a-SN 过表达以后脊髓神经元出现形态异常, rno-miR-138 抑制剂组与其相似，而a-SN 干扰组和rno-miR-138 模拟物则出现相反情况。IF显示α-SN 和凋亡相关分子 Bax、capease-3 和 Bcl-2共定位，充分说明 a-SN 启动凋亡的发生。此外，RT-PCR和Western Blot显示α-SN 干扰和rno-miR-138 模拟物组的 CNTF mRNA 和蛋白表达明显升高；a-SN 过表达和rno-miR-138 抑制剂组的凋亡相关基因 Caspase-7 和 Caspase-9 在 mRNA 和蛋白质水平表达明显升高。体内研究表明，α-SN 干扰和rno-miR-138 模拟物组的BBB评分显示脊髓损伤大鼠的后肢运动功能恢复；电生理检测显示大鼠SCI后，波幅和潜伏期均测不到。α-SN 干扰和rno-miR-138 模拟物组则能测到MEP，只是其潜伏期有些延长。BDA 示踪技术显示α-SN 干扰和rno-miR-138 模拟物组大鼠脊髓损伤处神经纤维再生。再者，3’UTR 双荧光素酶报告显示rno-miR-138能够靶向结合α-SN。综上，本研究首次探索了 SCI 后脊髓内 α-SN 和 miR138 与脊髓可塑性的关系，并探讨了脊髓横断伤后脊α-SN 表达的改变受到miR-138 的调控，进而影响脊髓损伤后脊髓结构和功能的恢复。因此，本项目有重要的理论意义和临床指导价值，为SCI修复治疗及调控机制研究提供强有力的理论依据。