电针治疗大鼠坐骨神经损伤的miRNA调控机制

Peripheral nerve injury (PNI) is a clinical commonly encountered disease. Electroacupuncture has great advantages for PNI treatment. Our previous study found that the proliferation of Schwann cells (SC) and secretion of neurotrophic factors (NTFs) is enhanced, which play an important role in the electroacupuncture for repair of PNI, but the mechanism of electroacupuncture to promote SC proliferation and NTFs secretion is not yet clear. MicroRNA (miRNA) involved in a variety of biological processes such as cell growth and proliferation. In this study, rat sciatic nerve injury model is established, miRNA is separated from the damaged tissue, differentially expressed miRNAs are picked through gene chip technology screening before and after electroacupuncture treatment of PNI. The differentially expressed miRNAs are verified by RT-PC, and the target gene prediction and functional enrichment of target gene pathway of obvious differentially expressed miRNA are analysised. Through in vitro cell culture and the local damaged tissue injection of miRNA agonists and antagonists, the biological function and mechanism of the most obvious differentially expressed of miRNA are studied further, trying to clarify "acupuncture - SC - NTFs - nerve regeneration" relationship, and providing objective and theoretical basis for exploring a new target for the treatment of the PNI and popularizing clinical electroacupuncture treatment for PNI.

周围神经损伤(PNI)是临床的多发病和常见病。电针对PNI的治疗具有较大的优势。我们前期的研究发现，雪旺细胞(SC)增殖和神经营养因子(NTFs)分泌增强在电针修复PNI中发挥重要作用，但电针促进SC增殖和NTFs分泌的作用机制目前尚不清楚。研究发现,微小核糖核酸(miRNA)参与细胞生长和增殖等多种生物学过程。本研究构建大鼠坐骨神经损伤模型，分离损伤组织中miRNA，采用miRNA基因芯片技术筛选电针治疗前后PNI组织中差异性表达的miRNA，通过RT-PCR对其进行验证，并对差异表达明显的miRNA进行靶基因预测及靶基因功能富集的通路分析。通过体外细胞培养和损伤组织局部注射miRNA的激动剂和拮抗剂，对差异表达最明显的miRNA的生物学作用及机制进行深入研究，力图阐明"针刺-SC-NTFs-神经再生"之间的关系，为探索治疗PNI的新靶点和临床推广电针治疗PNI提供客观依据和理论基础。

外周神经损伤（peripheral nerve injury, PNI）是临床上的常见病症之一，其病程长，严重影响个人生活质量，增加社会负担，是临床医师和研究者普遍关注的热点之一。中医针灸在治疗神经损伤的过程中发挥重要作用。在PNI后修复过程中，miRNA的表达变化可调节一系列相关基因的表达，从而参与神经修复与再生。本实验以坐骨神经损伤（sciatic nerve injury, SNI）大鼠为模型，评价了电针对SNI后功能恢复的作用。电针组腓肠肌湿量、神经传导速度均优于模型组。电针不仅可以促进损伤模型大鼠神经轴突的恢复和雪旺细胞（Schwann cell, SC）增殖，而且促进神经营养因子（neurotrophic factors, NTFs）的表达上调，包括NGF、BDNF、GDNF。miRNA测序和分析进一步发现，电针可上调或下调SNI后神经组织和脊髓中多种miRNA的表达，提示miRNA差异表达可能参与电针促进神经再生的作用。结合测序结果和qRT-PCR验证，本研究发现，与模型组相比，电针组神经组织中miR-1b的表达显著下调。高表达miR-1b和敲低其靶基因NDRG3、BDNF、VEGF可以抑制SC的增殖、迁移，并促使其凋亡。在电针治疗的基础上，对神经损伤组织局部注射差异性表达最明显的miR-1b的激动剂和拮抗剂，观察其对神经再生、SC细胞增殖和NTFs表达的影响。miR-1b可调节损伤同侧脊髓和血清中NTFs（NGF、GDNF）的表达，并对坐骨神经功能起到较显著改变，因此，miR-1b和其靶基因NDRG3、BDNF、VEGF介导的信号机制参与了电针促进PNI修复的作用。本研究从形态和功能上揭示了电针在损伤神经修复过程中的效应机制并阐明了“针刺-miRNA-SC-NTFs-神经再生”之间的联系，为临床推广电针治疗PNI和探索治疗PNI的新靶点提供了实验依据和理论基础。