分娩性臂丛损伤手内肌肱二头肌神经肌接头调控机制的基础研究

Clinically in obstetric brachial plexus palsy (OBPP), the atrophy of the intrinsic musculature of the hand (IMH) turns irreversible much faster than that of muscles of the upper arm. In our previous studies of microarray screening for miRNAs and mRNAs in the rat model simulating OBPP where atrophy of IMH is irreversible while atrophy of the biceps is reversible, it was found that denervated intrinsic musculature of the forepaw (IMF) and biceps had their own specifically dysregulated miRNAs, respectively, as compared with control. Analysis of GO and of pathway showed that those specifically dysregulated miRNAs and their target genes might participate in self-regulation of neuromuscular junctions (NMJs). We also found that dysregulated mRNAs in IMF were associated with inactive pathways of muscle regulation, and in the biceps they were associated with active pathways of regrowth and differentiation. Considering that the structure and function of IMH is much more complex compared with that of the biceps, and theoretically the postnatal development of NMJ is more immature in IMH than in the biceps, we hypothesize that in infants and young children, denervated NMJs of IMH is more susceptible to the harmful influence of inflammation and apoptosis than those of the biceps, while in the later, NMJs have relatively mature self ― protective potential to counteract that adverse influence. And this may be the reason that the structure of NMJs can be maintained stably for longer time in the denervated biceps than in the denervated IMH in OBPP. We will make focused DNA microarray to sift genes that affect self-regulation of NMJs, from denervated IMF and denervated biceps by comparison of their respective normal contralateral counterparts. That microarray includes genes of pathways of inflammation, apoptosis, re-growth and differentiation, structural genes of NMJs and genes that participate in maintaining stability of NMJs. RT-qPCR is used to directly compare the expression of sifted dysregulated genes between denervated IMF and denervated biceps. The determinations in vitro and in vivo are applied to testify those gained “crucial” genes. The purpose of this study is to find and confirm the crucial genes that induce the structure of NMJs of IMF to be more inclined towards instability than that of the bicep in the rat model of OBPP where atrophy of IMH is irreversible while atrophy of the biceps is reversible.

分娩性臂丛损伤手内肌较上臂大肌肉更快发生不可逆萎缩。前期对大鼠模型miRNA和mRNA表达谱筛查发现，不可逆萎缩前肢爪内肌和可逆萎缩肱二头肌差异表达miRNA预测的靶基因，多数参与神经肌接头（NMJ）自我调节；前肢爪内肌再生分化信号通路基因表达较肱二头肌明显不活跃。鉴于此及理论上手内肌NMJ较肱二头肌的成熟晚等特点，假设：婴幼儿失神经手内肌NMJ更易受炎症凋亡影响而发生结构不稳定，肱二头肌NMJ有较成熟自我保护潜能，因而在相对长时间内平衡上述不利影响。将制作功能分类基因芯片筛选影响失神经NMJ自我调节基因，其包括炎症、凋亡、再生和分化信号通路基因、NMJ结构基因及参与NMJ稳定修复的调节基因；用RT-qPCR等比较所筛选基因在二种肌肉中表达差异；体外体内验证所得 “关键”基因。研究目标是确认在分娩性臂丛损伤大鼠模型中，导致失神经前肢爪内肌NMJ较肱二头肌NMJ更易发生结构不稳定的关键基因。

临床上，分娩性臂丛损伤的患儿失神经支配手内肌几乎像成人一样很快进入不可逆萎缩状态，而上臂大肌肉的萎缩进程则相对缓慢。本项目的研究目的，是筛选与确认在“分娩性臂丛损伤手内肌不可逆萎缩、肱二头肌可逆萎缩”的大鼠模型中，导致失神经前肢爪内肌神经肌接头较失神经肱二头肌的更快发生结构不稳定的关键基因，为临床上优化该损伤的诊断和治疗提供科学依据。研究分4部分：①分娩性臂丛损伤不可萎缩手内肌与可逆萎缩肱二头肌circRNA和蛋白组学表达特征；②分娩性臂丛损伤大鼠模型前肢爪内肌和肱二头肌运动终板形态学、以及Wnt3a/ β-catenin信号通路与失神经前肢爪内肌和肱二头肌乙酰胆碱受体亚单位的表达关系；③Neuregulin-1/ErbB通过mTOR调控分娩性臂丛损伤手内肌和肱二头肌运动终板的机制；④产瘫大鼠模型前肢爪内肌与肱二头肌终末雪旺细胞的形态学比较。结果发现：①相比失神经肱二头肌，前肢爪内肌在失神经后发生更明显的炎症凋亡反应，而再生分化作用较弱；动态分析显示该这些信号通路在三周时明显活跃，五周时显著减退，但肱二头肌在失神经萎缩过程中炎症凋亡通路始终不活跃。②相对肱二头肌，Wnt3a/ β-catenin在失神经前肢爪内肌的表达明显活跃；该信号通路抑制L6成肌细胞中Rapsyn和AChR、AChR、 AChR亚基表达，表明其通过下调Rapsyn表达以抑制 AChR聚集。③失神经前肢爪内肌和肱二头肌中NRG-1/ErbB4通路同AChR各亚单位的蛋白表达呈正相关，并且失神经前肢爪内肌中NRG-1/ErbB4通路及AChR各亚单位的表达较失神经肱二头肌的下降更为明显； NRG-1/ErbB4通过Akt/mTOR/p70S6K途径调控运动终板AChR各亚单位蛋白表达。④失神经前肢爪内肌中，终末雪旺细胞相对运动终板覆盖率较正常显著减少，而肱二头肌的无显著差别。这些结果至少部分地揭示了大鼠模型中，失神经前肢爪内肌运动终板等较肱二头肌的更早发生退行性变的分子机制，而这些因素促使前者更快进入不可逆萎缩状态。这些结果不仅为分娩性臂丛损伤患儿神经重建手术时机的优化、也为临床上筛选与确认失神经支配肌肉进入不可逆萎缩状态的生物学标志，提供了一定的理论依据。