基于Nogo-A/RhoA/ROCK通路研究强制性运动精准治疗偏瘫型脑瘫的作用与机制

Children with hemiplegic cerebral palsy mostly have the unilateral hand dysfunction, which can lead to the high rate of physical disabilities and restrict their daily life and educational activity. The effects of traditional rehabilitation therapy (e.g. occupational therapy, electrical stimulation and splinting) to improve hand dysfunction remain uncertain in children with hemiplegic cerebral palsy. However, most of the recent studies have reported positive results of constraint-induced movement therapy. The study indicated that constraint-induced movement therapy could regulate the expressions of Nogo-A and its receptor NR1 during the process of neural regeneration and remodeling. Recent studies suggest that constraint-induced movement therapy might induce not only functional reorganization, but also structural plasticity. However, the underlying mechanisms of constraint-induced movement therapy induced structural plasticity are still unclear, and remain to be studied in children with hemiplegic cerebral palsy. In previous studies we found that constraint-induced movement therapy could significantly improve the hand function in children with hemiplegic cerebral palsy. In addition, our preliminary experiment showed that Nogo-A and NR1 gene of peripheral blood were significantly higher in mice model and children with hemiplegic cerebral palsy, suggesting that Nogo-A play an important role in the development of hemiplegic cerebral palsy. Therefore, we hypothesize that constraint-induced movement therapy may inhibit Nogo-A/RhoA/Rock signaling pathway so as to promote the improvement of hand function in children with cerebral palsy. On the basis of the preliminary experiment, we plan to establish the animal model of hemiplegic cerebral palsy and apply Nogo-A-shRNA interference and constraint-induced movement therapy to explore the effects of Nogo-A/RhoA/ROCK signaling pathway on neurons, axons and synaptosomes in mice model. In last this study combines the clinical research so as to further verify the role and mechaniasm of Nogo-A/RhoA/ROCK signaling pathway in constraint-induced movement therapy for the treatment of hemiplegic cerebral palsy, thereby providing new ideas and theoretical basis for the precise treatment of hemiplegic cerebral palsy.

偏瘫型脑瘫（HCP）常伴手功能障碍，致残率极高；常规康复技术对其疗效不明确。目前已知强制性运动疗法（CIMT）可明显改善HCP手功能，且可通过调节Nogo-A及其受体NR1的表达参与神经再生及重塑，但其具体机制仍不清楚。前期研究我们证实CIMT疗效明确；近期预实验进一步发现HCP小鼠及患儿外周血Nogo-A及NR1基因明显增高，提示Nogo-A在HCP发生发展中可能起重要作用。据此我们推测CIMT 可通过抑制 Nogo-A 信号通路而促进 HCP手功能的改善。本项目拟在前期研究基础上，通过制备HCP模型小鼠，应用Nogo-A-shRNA干扰技术及CIMT治疗，探讨Nogo-A/RhoA/ROCK通路对HCP模型鼠及CIMT干预后神经元、轴突及突触体的影响；最后结合临床研究，进一步验证Nogo-A/RhoA/ROCK通路在CIMT治疗HCP中的调控作用，为精准治疗HCP提供新思路和科学依据。

偏瘫型脑瘫（HCP）常伴手功能障碍，致残率较高。目前研究表明强制性运动疗法（CIMT）可通过促进大脑神经重塑改善HCP手功能，但其具体调控机制不明。为此，本研究旨在明确CIMT可通过抑制Nogo-A信号通路进而促进HCP手功能的改善。本研究通过制备HCP模型小鼠，采用CIMT、非强制性运动疗法（UNCIMT）或Nogo-A-shRNA干扰技术进行干预，将实验小鼠分为Con、HCP、HCP+UNCIMT、HCP+CIMT、HCP+SN和HCP+SN+CIMT组。随后通过转棒仪测试、前臂悬吊试验、免疫组化、高尔基染色，透射电镜和蛋白质免疫印迹来检测各组小鼠运动功能变化、神经元和神经纤维密度、树突/轴突面积、髓鞘完整性和大脑运动皮层中Nogo-A/NgR/RhoA/ROCK蛋白的表达情况。研究结果发现，CIMT可显著改善HCP小鼠上肢的抓握能力及协调功能，增加神经元和神经纤维密度、树突/轴突面积，促进髓鞘完整性的结构修复，同时显著降低小鼠运动皮层Nogo-A/NgR/RhoA/ROCK蛋白表达，并且HCP小鼠运动功能与神经元、神经纤维密度正相关。本研究显示CIMT可通过抑制Nogo-A信号通路从而促进HCP手功能的改善。此外，本研究还进行了临床Nogo-A基因多态性与脑瘫关系的病例对照研究，临床收集病例数1192例（600例正常儿童和592例脑瘫儿童）。结果发现Nogo-A的SNP位点rs1012603和rs17046518与脑瘫显著相关；单倍型“TTGGG”与患脑瘫的风险增加显著相关（尤其在痉挛性双瘫中），它主要影响具有较好运动和社会功能的脑瘫儿童（GMFCS I级，ADL>9）。本项目明确了Nogo-A/NgR/RhoA/ROCK通路在CIMT治疗HCP中的作用，从Nogo-A角度解析了HCP手功能改善的关键调控靶点，不仅为CIMT治疗HCP增添新的理论依据，也为HCP的精准治疗提供了新思路。同时临床研究为脑瘫基因水平的机制研究提供了新的思路，为脑瘫的个性化治疗提供理论支持。