基于全息化时序性基因表达谱分析的脊髓损伤分子病理机制及干预研究

Spinal cord injury (SCI) has remained a challenging area for scientists and clinicians due to the adverse and complex nature of its pathogenesis. SCI induces a progressive pathophysiology affecting cell survival and neurological integrity via complex and evolving molecular cascades whose interrelationships are not fully understood. To recapitulate the molecular mechanism of SCI process is a critical issue in both research and treatment studies. Injuries in spinal cord tissue leads to a rapid destruction of cells at the site of injury, an intense inflammatory response, secondary necrotic and apoptotic cell death, and reparative responses. These responses to injury are likely to be mediated and reflected by changes in mRNA concentrations, regardless of whether these changes are due to regulated gene expression or to altered cellular populations. However, histologic methods and behavior evaluations which were common techniques in previous studies could not display the gene expression properties of SCI. Recently, transcriptome analysis provides a novel approach to demonstrate all the gene expression details during the SCI process. This spatial and temporal analysis is based on whole genome transcription and gene ontology analysis, leading to evaluate the dynamic alternation of all the gene expressions related to the variety of biological and pathological SCI process. In this study, we sought to define the temporal distribution of mRNA changes following contusion spinal cord injury. By clustering genes with similar expression patterns, mRNA changes that can predict or explain cellular and tissue responses to injury will be captured and then analyzed. Second, following the guide of expression pattern, mechanism-specific therapies with small molecules, neurotrophins and cell transplantations will be performed. With repeating the transcriptome analysis after treatments, traditional evaluations such as RT-PCR, immunohistologic staining and BMS score would be carried out to exam the effect of transcriptome-guided therapies. Third, altered transcriptome after therapies will be compared with the one without treatments by both dynamic and time-course assays. The detailed results of transcriptome allow to reveal unique functional molecular signatures in each particular mechanism progress and each post lesion time-point. Meanwhile, to demonstrate this highly regulated molecular network of SCI process, prolong comparison between all mechanisms related to SCI process could be carried out. Our study provides insights into coordinated patterns of gene expression within the injury and the interrelationships of neurodegenerative and neural repair processes after injury. It will consolidate the molecular fundament for both therapy and research of spinal cord injury in future.

脊髓损伤严重危害社会公共健康，阐明其分子病理机制是研究脊髓损伤及修复的关键科学问题。脊髓损伤是一个时序性、多机制相互影响的病理过程，具有时序性和全息化特点的表达谱分析技术在研究脊髓损伤分子病理机制上能够发挥揭示分子本质重要的作用。本课题前期工作使用基因表达谱技术，对损伤脊髓组织进行时序性全基因组表达谱分析，初步揭示损伤后某些病理机制相关基因转录水平的动态变化。并拟计划在本课题中予以更完整的基因表达谱分析和干预机制研究，不仅可以从分子学层面深入的探索脊髓损伤病理机制，还能为进一步干预治疗提供指导。然后基于表达谱分析结果，对重要病理机制进行特点病理时期的靶向性干预，再次从分子学角度评价干预后表达谱改变，同时辅以传统组织学、行为学评价，从本质上了解干预效果。最后，将干预前后的表达谱分析结果进行横向和纵向比较，探索脊髓损伤各种病理机制间的相互关系，为将来治疗和研究脊髓损伤建立分子学基础。

1.改进脊髓钳夹模型、背侧切割伤模型和横断模型的制作方法，建立横断模型，解决脊髓组织不能完全去除、术后动物死亡率高等问题，电生理、组织染色等方法验证模型稳定，为后续多种脊髓损伤实验提供稳定可靠的动物模型；.2.揭示构成胶质疤痕细胞的多种来源，其主要来源于固有星形胶质细胞激活、迁徙、分裂，少部分来源于室管膜细胞和NG2细胞，提示胶质瘢痕细胞可能存在不同亚群并具有复杂功能。.3.对小鼠脊髓损伤后第0、1、3、7、14、28、42天脊髓组织进行转录谱测序，WGCNA分析得到9个功能模块，并分析得出其中的关键基因，为后续干预研究提供了理论基础。.4.对小鼠脊髓损伤后第0、1、3、7、14、28、42天外周血进行转录谱测序，发现在损伤后第3天外周血中有神经相关基因一过性的高表达，外周血单个核细胞分离及染色发现其中存在NeuN+/Map2+/Cd45-的“神经元”，其意义有待进一步揭示。.5.揭示了中枢神经系统室管膜Cd133+细胞为内源性神经干细胞，并可被VEGF和bFGF激活，分化为神经元以及星形胶质细胞，为内源性神经干细胞修复脊髓损伤提供了理论基础。.6.在相同的脊髓损伤模型中，与野生型小鼠对比，免疫缺陷小鼠显示出更好的功能恢复。通过脊髓组织转录谱测序及WGCNA分析，表明低免疫状态状态有利于神经连接形成。.7.在大鼠全切损伤脊髓损伤模型中植入包含NT3的壳聚糖材料管，发现在其损伤区域有神经再生，且动物下肢功能显著恢复。转录谱测序揭示该材料具有抑制炎症、促进神经再生和血管再生的功能。.8.在小鼠全切损伤脊髓损伤模型中植入LDH-NT3材料，6周后在小鼠的损伤部位中清楚地观察到新生神经元和轴突。.9.证实DNMT3B1基因功能缺陷，在分化中期对于抑制神经发育基因的沉默作用并不完全，为神经干细胞干预提供理论基础。.10.移植过表达BDNF的人脐带间充质干细胞治疗小鼠脊髓损伤，发现其可以减轻局部炎性反应，抑制神经元凋亡，减少脱髓鞘现象及胶质瘢痕形成，从而达到一定的治疗效果。.11.通用丙戊酸预处理的骨髓间充质干细胞，高表达细胞趋化因子CXCR4，使其更容易在脊髓损伤部位富集从而治疗脊髓损伤。.12.发现淫羊藿苷能够缓解细胞氧化应激与炎症反应；减轻了脊髓组织的充血、水肿；激活了PI3K/AKT信号通路，显著抑制了线粒体及内质网凋亡通路，从而抑制了脊髓损伤后神经元的凋亡。