基于生长锥内外信号通路交汇作用的复健片促进脑梗死皮质脊髓束重塑机制研究

Corticospinal tract(CST) plasticity can promote motor function recovery following cerebral ischemia, but the mechanism is unclear. Present research demonstrated that Fujian tablet with the effect of tonifying the liver and kidney can promote CST plasticity of MCAO rats. Based on these results and the relation of the liver, kidney, sui and brain in TCM, the research is designed to further investigate the initial mechanism of CST plasticity and the relative effects of Fujian tablet on the cervical spinal cord of MCAO rats by using in situ hybridation to observe the gene expression of BDNF and NGF, ELISA to observe the content of cAMP and RT-qPCR to measure gene expression of GAP-43 and PKA. And the research is also to investigate the mechenism to regulate CST fibers to cross the midline of the spinal cord by using in situ hybridation and histochemistry to observe expression of slit-robo and Nogo-NgR in cervical spinal cord of model rats and analyzing the interaction between signaling pathways of slit-robo, Nogo-NgR and cAMP-PKA. So it can reveal the systematic mechansim of corticospinal tract plasticity in cervical spinal cord following cerebral ischemia, demonstrate promising specific targets of Fujian tablet outside the brain and provide a new support to clinical and experimental research of characteristic effect of tonifying the liver and kidney to promote motor function recovery following cerebral ischemia.

皮质脊髓束（CST）重塑可以有效促进局灶性脑梗死大鼠运动功能恢复，但机制尚不清楚。本课题拟在前期"滋补肝肾方复健片可促进MCAO大鼠CST重塑"的基础上，以"泛髓关系"的中医理论为指导，以皮质脊髓束重塑为切入点，通过原位杂交检测复健片干预后MCAO大鼠不同时间点颈髓BDNF和NGF基因表达，ELISA法检测颈髓cAMP含量，实时荧光定量PCR（RT-qPCR）检测颈髓GAP-43及PKA基因表达，从生长锥内、外因素探讨MCAO大鼠CST重塑启动及药物干预机制；原位杂交及免疫组化法观测复健片干预下模型大鼠颈髓slit-robo及Nogo-NgR基因和蛋白表达变化，分析其与cAMP-PKA信号通路之间的交汇作用，探讨复健片对皮质脊髓束纤维中线跨越及跨越后稳定生长的调控机制，以期系统揭示脑梗死颈髓水平皮质脊髓束发生重塑的机制，明确复健片的脑外作用靶点，为揭示复健片的作用机制奠定坚实的理论基础。

本课题基于“泛髓关系”假说，在前期课题“滋补肝肾方复健片可促进局灶性脑梗死大鼠皮质脊髓束重塑”的基础上，突破传统以脑组织为核心的神经修复机制研究思路，采用Western-Blot、荧光定量PCR检测模型大鼠失神经支配侧（左侧）颈髓不同时间点GAP-43表达，发现模型组GAP-43在术后2~4周末较之假手术组呈高表达，增高趋势至术后4周末消失，BWT评分证实模型大鼠受损的运动功能也相应改善，表明失神经支配侧颈髓层面的神经纤维重塑可能起止于术后2~4周末，药物组GAP-43的表达则在术后3~5周末显著高于模型组和假手术组，提示复健片可持续增强MCAO大鼠颈髓层面神经纤维的重塑；采用Western-Blot、免疫荧光法检测模型大鼠颈髓不同时间点BDNF、NGF蛋白表达变化，免疫荧光法检测颈髓Nogo-A、NgR， ELISA法检测颈髓cAMP表达变化，RT-qPCR检测颈髓PKA-C mRNA，发现术后1周、2周末，模型组左侧颈髓的BDNF、NGF及颈髓cAMP、PKA-C的表达均高于假手术组，Nogo-A和NgR表达则低于假手术组，术后2周末，药物组上述指标变化的趋势更为明显，提示脑梗死后，颈髓可能通过高表达BDNF、NGF，上调cAMP、PKA-C水平，低表达Nogo-A、NgR，以促进神经轴突重塑，启动脑梗死后皮质脊髓束中线跨越，而复健片则可进一步上调脑梗死后颈髓BDNF、NGF表达和cAMP、PKA-C含量，抑制颈髓Nogo-A、NgR表达,以促进脑梗死皮质脊髓束中线跨越重塑的启动；采用BDA顺行神经示踪法明确MCAO大鼠皮质脊髓束中线跨越主要集中在颈髓后索近灰质处，进而分别采用免疫荧光法和Western-Blot检测右侧颈髓近中线白质区域Slit、Robo各蛋白表达及其下游通路Rac1、Cdc42、RhoA蛋白表达变化，结果表明，术后2~3周末，较之假手术组，模型组和药物组大鼠的Slit1、Slit2、Rac1及Cdc42蛋白表达增高，Robo1、Robo2、Robo3、RhoA蛋白表达降低，药物组变化趋势更为显著，并延续到术后4周末。提示脑梗死后颈髓Robo受体蛋白表达的下调有助于皮质脊髓束的中线跨越，而复健片则可通过进一步降低Robo受体表达，这可能是复健片促进皮质脊髓束中线跨越的重要机制之一。