【作者】 朱慧；

【导师】 顾晓松；

【作者基本信息】 南京大学 ， 生物学， 2017， 博士

【摘要】 周围神经损伤后的修复与再生是一个极其复杂的过程,成功的神经再生除了需要神经元内在生长力的激活,还需要建立一个良好的再生微环境,包括施万细胞的大量增殖、多种神经营养因子的参与和损伤局部良好的血液供应等。其中,神经细胞间是否能够形成有效的细胞间通讯是周围神经损伤后再生的关键。隧道纳米管(Tunneling nanotubes,TNTs)是近年来发现的新型动物细胞间的连接方式。TNTs是一类以肌动蛋白为基础成分的细胞膜性管道,广泛地存在于培养的细胞和重要的器官组织中,具有重要的生理学意义。TNTs可以运输多种细胞成分,并且通过传递电信号、运输细胞器来高效地介导细胞间的通讯。目前对周围神经系统是否存在TNTs及其在周围神经损伤再生过程的意义尚不清楚。因此,本文通过体外培养的施万细胞及大鼠坐骨神经离断模型对TNTs进行了系统的研究。研究发现体外培养的施万细胞中确实存在TNTs,同时我们检测到神经营养因子、膜蛋白、细胞质蛋白、线粒体和RNA在功能性TNTs中的转运。对TNTs的形成条件进行研究,由于细胞应激对TNTs的形成具有促进作用,结果表明,血清剥夺模型引起的细胞应激会诱导施万细胞中TNTs的大量形成,并且可以传递给非应激状态的受体细胞。大鼠坐骨神经损伤后细胞处于缺血缺氧的应激状态,在神经损伤近端可以观察到大量TNTs的形成。进一步对其形成的分子机制研究发现,TNTs的形成伴随施万细胞的迁移和线粒体能量的运输,同时Rab8a/Rab11a参与了TNTs中营养因子BDNF的转运,干扰Rab8a/Rab11a的表达导致施万细胞中TNTs的形成减少、BDNF分泌受抑制,迁移能力受抑制,同时促进细胞凋亡。在体外施万细胞与背根神经节(dorsal root ganglia,DRG)神经元共培养模型中,干扰Rab8a/Rab11a的表达会抑制神经元突起的生长。结果提示,Rab8a/Rab11a可能参与了周围神经系统中TNTs结构的形成,这种结构通过调控神经细胞之间的通讯最终影响了周围神经损伤后的再生过程。施万细胞是周围神经系统中的胶质细胞,可以分泌多种神经营养因子、粘附分子、细胞外基质分子,形成周围神经再生的微环境,支持和引导神经轴突的生长。胰岛素样生长因子-1(Insulin-like growth factor-1,IGF-1)作为重要的神经营养因子,能显著促进周围神经系统损伤后的轴突再生。采用大鼠坐骨神经离断模型,结合转录组测序及生物信息学软件分析,发现大鼠坐骨神经离断后,L4-L6背根神经节组织及损伤近端神经中IGF-1的表达水平升高,且损伤近端更为显著,提示损伤处积聚的IGF-1主要来源于受损的施万细胞。双报告基因系统检测结果表明,miR-129直接靶向IGF-1 3’-UTR来抑制IGF-1的表达。在坐骨神经再生过程中miR-129的表达与IGF-1的表达基本呈现负相关关系。从自分泌角度而言,miR-129/IGF-1调控途径一方面影响DRG神经元的突起生长,另一方面调控施万细胞的增殖、迁移和IGF-1的分泌功能。构建神经元和施万细胞共培养模型,研究miR-129对IGF-1及其受体的调控作用。结果从旁分泌角度阐明miR-129可以通过调控施万细胞IGF-1分泌进而影响神经元突起的生长。综上所述,本文首次探讨了 miR-129在周围神经损伤再生过程中对IGF-1转录后调控作用,丰富了 miRNA在神经系统疾病中的研究,有助于研究与开发miR-129作为新的治疗靶点,为优化和完善周围神经损伤的靶向治疗提供了科学依据。更多还原

【Abstract】 Peripheral nerve regeneration is a complex process orchestrating transcriptional and translational events at the level of the cell body,local translation at the regenerating nerve stump,interactions between outgrowing processes and microenvironment,and responsiveness of target organs.Successful nerve regeneration requires the establishment of a favorable regenerative microenvironment,including activation of viable Schwann cells(SCs),production of various neurotrophic factors and local well blood supply.Among them,effective intercellular communication of the neural cells is crucial to the peripheral nerve regeneration.Tunneling nanotubes(TNTs)are F-actin-based membrane tubes,and can form between cultured cells and within vital tissues.TNTs mediate intercellular communications that range from electrical signaling to the transfer of organelles.Following PNI,the orchestrated intercellular communications among neural and non-neural cells are required for effective nerve regeneration.It remains unknown whether TNTs exist between neural cells in the peripheral nerve system and how TNTs affect neural regeneration.To address these interesting questions,we investigated the transfer of neurotropic factors,membrane protein,cytoplasmic protein,mitochondria and RNA in functional TNTs formed between cultured SCs.TNT-like structures were increased not only in cultured SCs after exposure to serum depletion but also in longitudinal sections of proximal sciatic nerve stump harvested after rat peripheral nerve transection.Meanwhile,down-regulation of Rab8a or Rab11a in cultured SCs inhibited the formation of functional TNTs and vesicle transfer and led to decrease in cell migration,increase in SCs apoptosis.Likewise,knockdown of Rab8a or Rab11a in primary SCs also suppressed axonal outgrowth from co-cultured dorsal root ganglions(DRG)neurons.Overall,our results suggested that the gene of Rab8a or Rabl la might be involved in the formation of TNTs structures in the peripheral nerve system,while TNTs structures were likely to affect peripheral nerve regeneration through the regulation of neural cell communications.Moreover,following PNI the molecular mechanism on regulation of regeneration microenvironment and the effect on the intrinsic growth ability of neurons are very important.SCs are unique glial cells in the PNS and may secrete multiple neurotrophic factors,adhesion molecules,extracellular matrix molecules to form the microenvironment of peripheral nerve regeneration,guiding and supporting axonal outgrowth.Among them,insulin-like growth factor-1(IGF-1),as one of the important neurotrophic factors,can significantly promote axonal regeneration after PNI.We previously identified a group of novel miRNAs in proximal nerve following rat sciatic nerve transection by a combination with the transcription level high throughput data and bioinformatics software prediction,we predicted and validated the upstream miRNA regulating IGF-1.The result showed the increased expression level of IGF-1 in L4-L6 dorsal root ganglions and proximal nerve stump,which is more significant following sciatic nerve transection.It suggested that IGF-1 secretion mainly from SCs in PNI.The present study investigated the role of miR-129 in the proliferation and migration of SCs after sciatic nerve injury.An increased expression of miR-129 inhibited cell proliferation and migration of SCs,and inversely,silencing of the miR-129 expression promoted cell proliferation and migration of SCs.The IGF-1 was identified as one of the multiple targets of miR-129,which exerted negative regulation of IGF-1 by mRNA degradation.The temporal change profile of the miR-129 expression was negatively correlated with that of the IGF-1 expression in proximal nerve following sciatic nerve transection.Moreover,knockdown of IGF-1 attenuated the promoting effects of miR-129 inhibitor on SC prolifetration and migration.Overall,our data indicate that miR-129 affects phenotype modulation of SCs by targeting IGF-1,providing further insights into the regulatory role of miRNAs in peripheral nerve regeneration.In summary,the time-dependent expression profiles of miR-129 in the injured nerve were observed following peripheral nerve transection.SCs proliferation and migration were specifically regulated by miR-129 through targeting IGF-1 in vitro and in vivo.The downregulation of miR-129 stimulated SCs to increase IGF-1 production,which further encouraged axon regrowth.Accordingly,this study not only provides new insight into miR-129 regulation of peripheral nerve regeneration by robust phenotypic modulation of neural cells,but also opens a novel therapeutic window for PNI by mediating IGF-1 production.Our results may provide further experimental basis for translation of the molecular therapy into the clinic.更多还原