激活态OECs协同SSCs源性神经元促脊髓损伤修复的作用及分子机制

Neuronal degeneration, necrosis, and further neurological deterioration of the microenvironment following traumatic spinal cord injury (SCI) have become a significant bottleneck in neural regeneration and functional recovery. Until now, no treatment has shown efficacy. Therefore, it is pivotal solution to seek promising alternative cell sources and effectively improve host nerve injurious microenvironment for neural repair and functional recovery. Fascinatingly, our recent studies evidenced that the activated olfactory ensheathing cells (OECs) exhibited a remarkably increased proliferative activity, improved neurotrophic factors-secreting capacity, and potentiated phagocytosis activity. In addition, OECs also effectively promote transdifferentiation of spermatogonial stem cells (SSCs) to spinal cord neural cells under involvement of a set of defined growth factors. On the basis of the results obtained from previous project, the aims of our present study are to systematically investigate the role of activated OECs in improving microenvironment of spinal cord injury, promoting transdifferentiation of rat SSCs to spinal cord neurons and maintenance of microenvironment stability. Meanwhile, the characteristics of spinal cord neurons derived SSCs were identified by using the morphological, cellular and biochemical assays. We will eventually develop an efficient technology platform for both transdifferentiation of SSCs to spinal cord neurons and improving host intrinsic nerve injury microenvironment by the activated OEC transplantation, which could contribute immensely to clinical application of SSCs as a novel alternative stem cell source and tissue engineering approaches for spinal cord injury (SCI). Furthermore, up till now an effort still remains in understanding of the underlying molecular mechanisms regarding how the activated OECs involve in SSCs differentiation and further its survival. Thus the present study will further elucidate the molecular mechanisms by which activated OECs improve injurious microenvironment and promote SSCs-derived neurons through Akt/NF-κB signaling pathways. It may provide novel theoretical basis for clarification of the molecular mechanism in future clinical application.

脊髓损伤（SCI）引起神经元退变坏死及损伤微环境逐步恶化已成为其再生和功能恢复的瓶颈，目前尚无有效的治疗手段。因此寻求理想的替代细胞并有效改善损伤微环境对神经修复和功能恢复尤为重要。我们前期研究发现，嗅鞘细胞（OEC）激活后其增殖力、分泌神经营养因子及清除坏死神经碎片的能力显著提高，并能联合限定性因子诱导精原干细胞（SSC）分化成脊髓神经元。本项目拟在先前课题基础上以激活态OEC和SSC为切入点，利用细胞分子生物学等技术深入探讨激活态OEC对损伤微环境的改善及对SSC诱导产生神经元的保护作用。拟建立有效改善SCI微环境并进一步提高移植细胞存活的新技术平台，为临床SCI的细胞治疗和组织工程提供重要的细胞来源和新的思路。目前OEC激活后如何影响神经元存活机制尚不清楚，本项目将阐明激活态OEC改善微环境促进SSC源性神经元存活的Akt/NF-κB信号转导通路，为临床上治疗SCI提供新的分子机制。

脊髓损伤（SCI）是一种致残率和致死率极高的中枢神经系统(CNS)创伤性疾病，也是困扰临床工作者的棘手难题。SCI发生后进行性的动态病理变化引起神经元变性、坏死是最终导致不可逆系统功能丧失及机体功能障碍的主要原因，迄今尚无有效的治疗手段，因此寻求切实有效的治疗方案迫在眉睫。神经干细胞（NSCs）具有独特的优势，然而由于NSCs来源严重匮乏以及伦理等诸多问题使其在临床上应用仍面临着巨大挑战。因此寻求理想的替代细胞显得尤为重要。我们先前研究发现，精原干细胞（SSCs）在条件培养基诱导下可以分化为脊髓神经元，且SSCs分化产生的神经元无需基因修饰等复杂程序，与其他种类干细胞相比具有来源容易（SSCs动物终生不断产生）、无伦理问题、无致瘤性和免疫排斥等优势。SCI发生后，微环境呈现进行性恶化严重制约了受损神经元再生，不利于细胞的存活，因此本项目拟以激活态OECs（aOECs）和SSCs为切入点，利用细胞分子生物学等技术探讨aOECs对损伤微环境的改善及对SSCs诱导产生神经元的保护作用。进一步阐明aOEC改善微环境并促进SSC源性神经元存活的信号通路，最终建立高效移植细胞存活的新技术平台，为临床SCI的细胞治疗提供重要的细胞来源和新思路。我们研究结果显示：LPS联合CCM诱导OECs激活，aOECs增殖能力显著增强，分泌产生的神经营养因子（BDNF、NGF和GDNF）抗炎性因子（IL-4、IL-10和TGFβ）均进一步显著提高（*p<0．05, **p<0．01），且吞噬凋亡的神经细胞碎片的能力也显著增强；aOECs在炎性损伤退变神经碎片介入条件下可促进神经元存活和突起生长。统计学分析显示：aOECs体系诱导SSCs产生的不同亚型神经元比例上显著高于单纯OECs诱导（*p<0．05, **p<0．01），而分化产生的神经元更具有野生型神经元的特征如突触形成、电生理活动及神经元钙成像。将aOECs和SSCs移植到受损的脊髓中，结果发现移植aOECs和SSCs后，脊髓完整性明显好于单纯的OECs组及对照组，行为学和神经功能评估也表明，联合移植组的功能恢复明显好于其他组。aOECs诱导SSCs向脊髓神经元分化并促进脊髓功能恢复可能与Akt/NF-κB信号通路靶向下游分子调控上调神经营养/抗炎性因子密切相关。因此本研究有望为临床SCI的细胞治疗提供重要的细胞来源和新思路。