PirB蛋白介导神经抑制因子-整合素αv信号在视神经再生中的作用与机制研究

Myelin-associated inhibitors in central microenvironment play a vital role in the failure of axons regeneration of adult mammalian central neurons. Previous studies have shown three major myelin inhibitors: the C-terminal of Nogo-A (Nogo-66), myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), which all interacted with the same receptor NgR to inhibit axonal regeneration. However, many studies, including our previous work, have revealed that the regenerate ability of central neurons was still limited with the absence of NgR.In recent issues of ''Science'' and ''Neuron'', a second receptor for these inhibitors has been described, PirB, to which the three major inhibitors can also strongly bind to inhibit axonal regeneration. Although in vivo PirB seems not to play a role in inhibiting axonal regeneration following TBI and SCI, a recent study by Fujita et al. has surprisingly revealed that in vivo transfection of small interfering RNA (siRNA) for Src homology 2-containing protein tyrosine phosphatase (SHP)-1 and SHP-2 enhanced axonal regeneration after optic nerve injury in mice.Our recent pre-test also revealed that PirB was probably another key molecule in inhibition of optic nerve regeneration. Accordingly, based on previous study, in this project we would study the optic nerve regeneration effect in vivo and in vitro by inhibiting PirB and/or NgR function in association with the application of neurotrophin, and clarify the role and mechanism of PirB mediated inhibitor-integrin αv pathway in optic nerve regeneration. This project would provide experimental basis for regulating PirB protein to promote optic nerve regeneration, and lay the foundation for discovery and development of new efficient optic nerve regeneration drug.

中枢微环境中的神经生长抑制因子是视神经再生困难的重要原因。以往研究认为三种主要抑制因子Nogo-66、MAG、OMgp主要是通过共同受体NgR发挥抑制作用。然而我们前期研究表明阻抑NgR视神经再生仍然有限。近年研究发现,三种抑制因子还能通过另一共同受体PirB抑制轴突生长, 另外,最新研究证实:调节PirB信号通路下游关键分子SHP-1/SHP-2可明显促进视神经再生，说明PirB可能是视神经再生困难的又一重要原因。据此,本项目拟在前期工作基础上,通过体内和体外试验对比研究，分别阻抑PirB和NgR及联合阻抑PirB和NgR以及联合运用神经营养因子的促视神经再生效应,观察PirB下游整合素信号αv/FAK的变化，以期阐明PirB蛋白在视神经再生中的作用及与NgR完全不同的分子机制,为调节PirB蛋白和NgR达到促进视神经再生提供实验依据,并为探索新的高效促视神经再生方案奠定基础。

成年哺乳动物中枢神经系统神经损伤后修复十分困难，常导致严重的持续性神经功能障碍。许多眼外伤和致盲性眼病都是因为视神经发生不可逆的损害导致的，促进损伤视神经有效再生和功能恢复，帮助患者重见光明，是我国防盲治盲工作的重要组成部分，其任务艰巨，意义重大。中枢微环境中的神经生长抑制因子是视神经再生困难的重要原因。以往研究认为三种主要抑制因子 Nogo-66、MAG、OMgp主要是通过共同受体NgR发挥抑制作用。然而我们前期研究表明阻抑NgR视神经再生仍然有限。近年研究发现，三种抑制因子还能通过另一共同受体PirB 抑制轴突生长, 另外，最新研究证实：调节 PirB信号通路下游关键分子SHP-1/SHP-2可明显促进视神经再生，说明PirB可能是视神经再生困难的又一重要原因。据此，本项目在前期工作基础上，以体外原代培养的视网膜神经节细胞（RGCs）为对象，主要进行了如下3方面的研究：1.观察PirB在RGCs生长不同时间点的变化规律；2.采用单独阻抑PirB、NgR，以及联合运用神经营养因子策略，观察不同处理方式对于RGCs的促生长效应；3. 观察整合素信号分子αv在RGCs中的变化情况。主要结论为：1.发现PirB 在RGCs表达水平随着时间呈现先升高后下降的变化，其中第五天表达水平最高；2.通过慢病毒载体携带siRNA成功阻抑PirB和NgR表达，PirB阻断后RGCs轴突生长较对照组增强，联用CNTF更有助于改善RGCs生长状态，而NgR阻断后RGCs生长与对照相比无明显差异；3.发现整合素信号分子αv在RGCs中虽存在表达，但其与PirB变化不一致，提示在RGCs损伤修复中pirB介导的信号通路可能并非整合素信号αv信号通路。本项目为调节PirB蛋白达到促进视神经再生提供实验依据,为探索新的视神经再生方案奠定了基础。.本研究已按计划完成课题内容（部分调整项目见原因说明），培养研究生1名，暂未发表SCI论文。