Ahmed Zubair, Dent Russell G, Suggate Ellen L, Barrett Lee B, Seabright Ruth J, Berry Martin, Logan Ann
Molecular Neuroscience Group, Department of Medicine, University of Birmingham, 3rd Floor Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH, UK.
Mol Cell Neurosci. 2005 Mar;28(3):509-23. doi: 10.1016/j.mcn.2004.11.002.
The presence of multiple axon growth inhibitors may partly explain why central nervous system axons are generally incapable of regenerating after injury. Using RNA interference (RNAi) in dorsal root ganglia neurons (DRGN), we demonstrate siRNA-mediated silencing of components of the inhibitory signalling cascade, including p75NTR, NgR and Rho-A mRNA, of 70%, 100% and 100% of the relevant protein, respectively, while changes in neither protein levels nor cellular immunoreactivity were detected using the relevant scrambled siRNA control sequences. Importantly, after 48 h in culture after siRNA-mediated knockdown of Rho-A, neurite outgrowth was enhanced by 30% compared to that after p75NTR and 50% after NgR silencing. By 3 days, a 5-, 3.5- and 6.5-fold increase in betaIII-tubulin protein levels were observed compared to controls without siRNA after knockdown of p75NTR, NgR and Rho-A, respectively. Together, these results suggest that Rho-A knockdown might be the most effective target for a disinhibition strategy to promote CNS axon regeneration in vivo.
多种轴突生长抑制因子的存在可能部分解释了为什么中枢神经系统轴突在损伤后通常无法再生。我们在背根神经节神经元（DRGN）中使用RNA干扰（RNAi）技术，分别将抑制信号级联反应的成分（包括p75NTR、NgR和Rho-A mRNA）的相关蛋白沉默了70%、100%和100%，而使用相关的乱序siRNA对照序列时，未检测到蛋白质水平或细胞免疫反应性的变化。重要的是，在siRNA介导的Rho-A敲低后培养48小时，与p75NTR敲低后相比，神经突生长增强了30%，与NgR敲低后相比增强了50%。到第3天，在分别敲低p75NTR、NgR和Rho-A后，与未使用siRNA的对照相比，βIII-微管蛋白水平分别增加了5倍、3.5倍和6.5倍。总之，这些结果表明，敲低Rho-A可能是促进中枢神经系统轴突在体内再生的去抑制策略中最有效的靶点。
