GDNF预处理的感觉神经移植治疗臂丛神经根性撕脱伤的实验研究

Root avulsion of brachial plexus is a common while severe nerve injury. Direct re-implantation of ventral root as repair has been studied in basic science research, however, this method is difficult to apply in clinical settings. Transplantation of sensory nerve is more appropriate in clinical practice, however, the ability of Schwann cells (SCs) of sensory nerve to promote axonal regeneration of motor-neuron is weaker than SCs of motor nerve. Previous reports and our pilot studies have all shown that SCs from the GDNF pre-treated sensory nerve could promote the axonal regeneration of motor-neurons. Taken these together, we hypothesize that GDNF could exert functions on SCs via the MEK/ERK pathway, and subsequently induce SCs to express motor phenotypic markers as well as endogenous GDNF. Furthermore, transplantation of GDNF pre-treated sensory nerve to repair the root avulsion of brachial plexus could promote the axonal regeneration of motor-neurons. To test this hypothesis, we will perform both cell culture study and in-vivo animal study to investigate the mechanism of GDNF in modulating the SCs of sensory nerve. Furthermore, we will transplant the GDNF pre-treated sensory nerve to repair the root avulsion of brachial plexus to see its therapeutic effect. This project will elucidate the feasibility as well as the mechanism of transplanting GDNF pre-pretreated sensory nerve for the treatment of root avulsion of brachial plexus, and thus provides theoretical basis and therapeutic strategies for brachial plexus injury in clinical practice.

臂丛神经根性撕脱伤是一种临床常见严重神经损伤。实验研究中采用腹根直接再植修复，较难应用于临床。采用感觉神经移植的方法贴近临床实际情况，但感觉神经内的雪旺细胞对运动神经元轴突再生的支持能力较运动神经雪旺细胞弱。文献及申请者前期工作显示，经GDNF预处理的感觉神经雪旺细胞，促进运动神经元轴突生长的能力增强。我们推测：该效应是GDNF通过MEK/ERK通路作用于感觉神经雪旺细胞，使其更多表达运动表型标记物及内源性GDNF而达到的。经GDNF预处理的感觉神经移植修复臂丛根性撕脱伤，可促进运动神经元轴突再生。为验证这一假设，本课题拟通过细胞培养和体内实验，探索GDNF调控感觉神经雪旺细胞的机制，并采用GDNF预处理感觉神经延期桥接修复臂丛根性撕脱伤，观察其疗效。本课题将阐明GDNF预处理感觉神经作为自体神经移植物的可行性及相关机制，为临床上治疗臂丛神经根性撕脱伤提供理论依据与治疗策略。

目的:研究胶质细胞源性神经营养因子（GDNF）对SCs表型、基因表达和神经营养因子变化的影响。探讨移植胚胎神经元对大鼠周围神经损伤(PNI)模型远端神经残端雪旺细胞（SCs）的影响。.方法：实验一：用自组装肽纳米纤维支架(SAPNs)RADA-16I包裹GDNF于失神经支配的感觉神经断端。后采用预处理后的感觉神经移植修复运动神经损伤。比较术后运动轴突再生、髓鞘再生和肌肉功能恢复情况。qRT-PCR分析预处理感觉神经中在运动和感觉SCs中表达不同的基因的表达，探讨外源性GDNF应用后发生的变化。实验二：第一阶段切断右侧腓总神经(CPs)，将14天的胚胎(E14)神经元细胞或细胞培养液移植到CPs远端，并于术后6个月取出移植细胞(第二阶段手术-I)，比较移植部位远端SCs表型和神经营养因子表达模式变化。取一段移植部位远端的神经，桥接至新鲜切断的胫神经(TIB)(第二阶段手术-II)，检测是否具有促进轴突生长的作用。.结果：GDNF预处理组的BDNF表达分别是阴性对照（NC）组和VC组的2.3倍和2.5倍(P<0.05)。GDNF预处理组内源性GDNF的表达明显增强，分别是NC组和VC组的4.4倍和3.1倍(P<0.05)。GDNF预处理组和PC组的FG阳性神经元数目均明显高于NC组和VC组(P<0.05)。GDNF预处理组有髓神经纤维数量较NC组和VC组明显增多(P<0.05)。空白对照（VC）组的P75和S100表达在第一阶段和第二阶段手术-I后下降。在细胞移植组中，P75保持较低水平，S100保持较高水平，而移植细胞切除后P75和S100的表达出现逆转。细胞移植组第一阶段术后GAGs表达下调，髓鞘蛋白零(MPZ)的表达上调。第二阶段手术-I后两周，移植神经元切除后GAGs表达上调，MPZ表达下调。免疫组化染色(IHC)显示移植细胞组神经肌肉接头重建(NMJs)较多，神经再生率较高(P<0.05)。.结论：外源性GDNF直接作用于体内感觉神经移植物，增加内源性GDNF和感觉干细胞衍生标记物BDNF的表达，促进近端运动神经轴突的再生和髓鞘再生，促进二期手术后肌肉功能的恢复。移植到损伤神经远端的神经元可以与SCs相互作用，防止SCs的慢性变性和纤维化。神经元切除后再次激活SCs的能力为促进近端轴突的生长创造了有利的微环境。