CIBZ基因在神经干细胞移植靶向性修复小鼠脊髓损伤中的作用

Spinal cord injury (SCI) induces both primary uncontrollable mechanical injury and secondary controllable degeneration. Served as target pharmaceutical points, the vital role and activation of caspase-3 family proteins in apoptosis have been well demonstrated in animal models. Caspase-3 inhibitors have caught the attention of so many scientists for curing apoptosis-related diseases and for alleviating impairments induced by SCI. CIBZ, a novel Zinc finger and BTB domain-containing protein and reported negative regulator of apoptosis induced by capase-3 mitochondrial pathway, was discovered to implicate in the SCI process for the first time via mRNA differential display in mouse model. To explore its role in secondary injury and self-healing, lentiviral-mediated-shRNA and neural stem cells (NSCs ) transplantation methods are used in this study. After CIBZ overpression and CIBZ knockdown(-/-)modified NSCs are transplanted into the injury centre respectively, BBB score joint behavior, morphology of injury and regeneration（NSCs migration and differentiation etc）, action potential detection via single cell patchclamp and gene expression in mitochondrial pathway are used to assess the results of the genetic therapy and establish signal transduction of CIBZ gene in vivo and vitro. It will provide a promising way to deter the cell death-induced impairments and improve the functional recovery of SCI in the future.

脊髓继发性损伤导致严重的脊髓再生不良，通过线粒体途径活化caspase-3基因诱导细胞凋亡的过程是其中的关键环节，所以caspase-3是治疗脊髓损伤的重要靶点。本研究组通过对损伤后脊髓的差异基因表达谱分析,首次发现一种能抑制caspase-3活化的BTB结构域锌指蛋白CIBZ参与小鼠脊髓损伤进程。为了阐明CIBZ在继发性脊髓损伤和修复中的作用，本课题通过慢病毒介导shRNA及神经干细胞（NSCs）移植技术，将过表达和knockdown CIBZ的NSCs分别移植至损伤中心，对移植后动物进行BBB联合行为学评分、研究修复过程中NSCs的迁移和分化规律、单细胞膜片钳测定动作电位、分析线粒体凋亡通路相关基因的表达，最后评定移植后的效果，建立CIBZ在脊髓损伤和修复中的体内、外信号通路。该研究将为探索脊髓再生和功能恢复的分子机制提供新的思路，为治疗研究开辟新途径。

脊髓损伤是常见的中枢神经系统疾病，脊髓神经元损伤后难以再生，因此抑制早期神经元的死亡和促进神经元的再生是目前研究的热点。本课题组前期通过差异基因表达谱分析，首次发现一种能抑制caspase-3活化的BTB结构域锌指蛋白CIBZ参与小鼠脊髓损伤进程。为了阐明CIBZ在继发性脊髓损伤和修复中的作用，本课题首先通过体外培养SH-SY5Y细胞、构建Thapsigargin(TG)诱导内质网应激模型、体外shRNA干扰CIBZ表达，结果表明内质网应激会下调CIBZ的表达，促进内质网应激介导的细胞凋亡。同样在小鼠脊髓损伤模型中，脊髓损伤引发的内质网应激会抑制CIBZ表达导致细胞凋亡。通过染色质免疫沉淀联合实时荧光定量PCR技术分析表明，在正常脊髓组织中ATF4激活CIBZ的转录表达，然而在损伤脊髓组织中这种激活作用会降低。其次，通过单细胞膜片钳测定动作电位和分子生物学分析，在小鼠脊髓损伤后腹腔注射牛磺熊去氧胆酸（TUDCA）显著上调CIBZ的表达，降低内质网应激、减少神经细胞凋亡。最后，通过慢病毒介导shRNA及骨髓间充质干细胞（BMSCs）移植技术，将过表达CIBZ的BMSCs移植至损伤中心，结果表明CIBZ过表达后明显的减少了脊髓损伤后细胞凋亡，降低脊髓的继发性损伤，运动功能也得到明显改善，说明CIBZ可以作为脊髓损伤治疗的靶点，恢复其表达能够促进损伤后脊髓功能的恢复。该研究将为探索脊髓再生和功能恢复的分子机制提供新的思路，为治疗研究开辟新途径。