慢病毒介导lhRNA沉默PTPσ及NgR基因修复脊髓损伤的研究

CSPGs and MAIFs, two important axonal growth inhibitors after spinal cord injury, interact with their receptors (PTPσ and NgR) and result in the inhibition on axon growth in a combined action. It is a key step for studies on recovery of spinal cord injury to block inhibitory effects of CSPGs and MAIFs simultaneously, and to elucidate the inhibitory mechanisms network of CSPGs and MAIFs on axon growth mediated by PTPσ and NgR. Making use of the previously acquired PTPσ-shRNA and NgR-shRNA, we have successfully constructed the lentivirus-mediated lhRNA with high efficiency that silence PTPσ and NgR simultaneously. Based on these preliminary achievements, this study aims to investigate the effects of simultaneous blockage of CSPGs and MAIFs on the axon regeneration and elongation, as well as the neurological recovery, and analyze the proteomic changes of neurons after simultaneous blockage of PTPσ and NgR using iTRAQ differential proteomics and bioinformatics to reveal the differential proteins for the specific analysis of the PTPσ and NgR-mediated inhibitory mechanisms of CSPGs and MAIFs on axon growth，and preliminarily clarify the mainly potential mechanisms network of axonal inhibition and repair. This study would lay the foundation for blockage of axonal growth inhibitors and targeted therapy after spinal cord injury.

脊髓损伤后轴突生长抑制因子CSPGs及MAIFs与受体PTPσ及NgR相互作用，共同决定轴突的抑制作用。同时阻断CSPGs及MAIFs的抑制作用并阐明PTPσ及NgR介导的CSPGs及MAIFs的轴突抑制机制网络是脊髓损伤修复研究的关键步骤。课题组前期利用已获得的高抑制效率PTPσ-shRNA及NgR-shRNA，成功地构建了慢病毒介导lhRNA双基因干扰系统同时沉默PTPσ及NgR基因，本研究拟在此基础上：（1）探索同时阻断CSPGs及MAIFs对神经修复的影响；（2）应用神经元iTRAQ差异蛋白质组学及生物信息学技术，分析PTPσ及NgR双基因沉默后神经元蛋白质表达的改变，明确差异蛋白质，针对性地解析PTPσ及NgR介导的CSPGs及MAIFs的轴突抑制机制网络，初步阐明可能的主要轴突抑制及修复机制，为轴突生长抑制因子的阻断治疗及脊髓损伤的靶向治疗奠定理论基础。

脊髓损伤是高发生率、高致残率的严重创伤性疾病。轴突生长抑制因子CSPGs及MAIFs在脊髓损伤后抑制轴突再生，PTPσ及NgR作为受体与CSPGs及MAIFs相互作用抑制轴突再生。而同时阻断CSPGs及MAIFs的作用对促进脊髓损伤后轴突再生的效果仍不明确，PTPσ及NgR介导的CSPGs及MAIFs的轴突抑制机制网络也有待进一步阐明。本课题通过构建慢病毒介导lhRNA双基因干扰系统沉默PTPσ及NgR基因，同时阻断CSPGs 及MAIFs的轴突抑制作用，通过检测体外CSPGs-MAIFs（Nogo A）混合基质上的神经元轴突再生能力以及体内实验中皮质脊髓束穿越脊髓损伤区域的情况，证实了同时阻断CSPGs及MAIFs的作用将促进脊髓损伤后神经轴突的再生；同时脊髓损伤大鼠模型的感觉及运动诱发电位分析提示同时沉默PTPσ及NgR基因明显的促进了脊髓损伤后感觉及运动功能的恢复。为进一步明确PTPσ及NgR介导的CSPGs及MAIFs的轴突抑制机制网络，我们利用iTRAQ技术，对神经元进行蛋白组学分析，再进一步对发现的差异蛋白质进行生物信息学分析并构建差异蛋白质互相作用网络，对主要的差异蛋白质进行验证，验证结果与生物信息学分析结果相同。综上所述，慢病毒介导lhRNA双基因干扰系统沉默PTPσ及NgR基因，同时阻断CSPGs 及MAIFs的轴突抑制作用，明显促进了神经元轴突的再生及脊髓损伤后感觉和运动功能的恢复；同时，PTPσ及NgR基因介导的轴突抑制机制网络的阐明为脊髓损伤修复提供了新的理论依据。