Sox8在脊柱损伤导致神经元缺血再灌注中的调控作用和机制研究

Neuron ischemia/reperfusion injury caused by spinal injuries plays a crucial role in the morbidity of disability. The most important mechanism of its progression is associated with ischemia/reperfusion of neurons. The molecular regulation mechanism of ischemia/reperfusion of neurons is a key question in the study of spinal cord injury. Sox family members are main transcription factors in the development and differentiation of neurons, however, its specific regulation mechanism is still unknown. The applicant has been working on the study of spinal cord ischemia/reperfusion injury. According to the preliminary research foundation, we used spinal cord ischemia/reperfusion injury mouse models and screened the expression of Sox family members in neurons. Results showed Sox8 expression increased significantly. Based on these data, we constructed conditional knockout mice of Sox8, and found that neuron specific Sox8 knockout mice developed more severe spinal cord ischemia/reperfusion injury, while Sox8 depletion in microglia or astrocyte has little effect. Therefore, we intend to investigate the regulatory mechanisms of Sox8 in the neuron ischemia/reperfusion injury, hoping to propose a new mechanism and an intervention approach of spinal cord ischemia reperfusion injury.

脊柱损伤导致神经元缺血再灌注是其致残的重要因素，神经元因缺血再灌注而坏死凋亡是脊髓损伤致残的重要机制，神经元缺血再灌注的分子调控机制是脊髓损伤研究中关键的科学问题。Sox家族分子是神经元分化发育中重要的转录因子，但Sox家族分子在神经元缺血再灌注中的调控作用目前尚不清楚。申请人长期从事脊髓缺血再灌注损伤的调控机制研究，根据前期基础，我们在脊髓神经元缺血再灌注疾病模型中，系统筛选了其基因表达谱改变，并重点关注Sox家族分子，发现Sox8发生了最为显著的表达上调。我们据此构建了Sox8的条件型基因敲除小鼠，发现神经元特异性Sox8敲除显著增强了小鼠脊髓缺血再灌注损伤疾病进展，而星型胶质细胞和小胶质细胞Sox8敲除无明显影响。因此，本研究拟探求Sox8调控脊髓缺血再灌损伤发生发展的作用和机制，尤其关注Sox8调控神经元缺血再灌注损伤的分子机制，以期提出脊髓缺血再灌注损伤发病新机制和干预新思路。

背景：脊柱外科中，急性创伤性脊柱外伤及长时间严重后纵韧带骨化后的解压手术往往会导致脊髓发生缺血再灌注损伤（SCIR），导致脊髓二次损伤。尽管目前人类已经进行了一些探索，但目前针对SCIR后脊髓的基因表达变化缺少系统性研究，尚不明确关键致病因子，对于脊髓的自身调节机制也缺乏了解。..主要研究内容：本研究采用临时主动脉夹闭再灌注法建立SCIR小鼠模型。采用mouse Basso/Beattie/Bresnahan scale(mBBB)评分评估小鼠运动功能。脊髓HE染色和TUNEL染色分析脊髓损伤区。进行高通量转录组测序以探索SCIR后基因表达改变并筛选潜在靶点，其中重点关注Sox8基因表达变化及高差异表达基因。用N2a细胞体外氧-葡萄糖-血清剥夺/恢复(OGSD/R)模拟SCIR的病理生理过程。..重要结果：高通量测序、RT-PCR和Western blot结果显示，SOX8在小鼠SCIR后无显著改变，与前期的体外实验结果不相符。通过进一步筛选高差异表达基因发现，Trem-1表达在小鼠SCIR后显著上调。进一步研究发现Trem-1与SYK相互作用，通过PI3K/AKT和NF-κB信号通路介导神经元凋亡和炎症反应。鞘内注射含有Trem-1 shRNA的腺病毒可抑制SCIRI引起的神经元凋亡。靶向Trem-1可能是治疗SCIR患者的候选治疗方法。..关键数据：脊髓缺血再灌注损伤后细胞凋亡增加；氧-葡萄糖-血清剥夺/恢复（OGSD/R）可于N2a细胞建立SCIR体外模型；SCIR后Trem-1的表达水平上调；Trem-1参与OGSD/R诱导的N2a细胞凋亡；Trem-1通过与SYK相互作用触发了PI3K/AKT和NF-κB信号通路；SCIR后Trem-1介导脊髓神经元凋亡，抑制Trem-1表达可减少凋亡发生。..科学意义：从理论上更新了对脊髓缺血再灌注损伤过程中相关信号通路和分子机制的理解，并进一步丰富对脊髓缺血再灌注损伤发病机制的认识，为研究脊髓缺血再灌注损伤提供理论基础，为临床预防和治疗脊髓缺血再灌注损伤提供了新方法、新思路。