Reg-2基因修饰的嗅鞘细胞移植在脊髓损伤中的神经修复机制

The restitution of the cut central connection of the pathway for movement control is the pivotal process for functional recovery of injured spinal cord. Olfactory ensheathing cells (OECs) transplantation following spinal cord injury (SCI), as a promising or potential therapy for spinal cord injury (SCI), was capable of promoting axonal outgrowth and remyelination. However, up to now, the underlying mechanism for the effects of transplanted OECs on the remodeling of injured corticospinal tract and neuronal regeneration is largely unknown. Based on our previous findings that Reg-2 presents a neuroprotective effect for treatment of SCI, the cultured OECs will be transduced with lentiviral vectors encoding Reg-2 gene after cloning of the Reg-2 gene, and transplanted into injured spinal cord in rat, thereafter, the effects of OECs on the axonal outgrowth of corticospinal tract will be estimated . We will focus on the putative effects of grafted OECs on axonal outgrowth from injured corticospinal tract. Our proposal will involve a combinatorial approach that integrates cloning of the Reg-2 gene, culture of OECs co-infected with lentivirus, use of rat SCI model, axon tract tracing, DTI imaging, behavioral test, and molecular assays, and investigate the effects of grafted OECs following SCI on neuronal survival/regeneration, sprouting of injured corticospinal axons along the disrupted axonal tract, then further elucidate the underlying basis for this, elucidate the underlying mechanism for the remodeling of corticospinal tract and neuronal regeneration after OECs transplantation. Moreover, our findings will provide theoretical basis for the search of novel drugs or potential therapeutic targets for treatment of spinal cord injury.

影响脊髓损伤后功能恢复的关键是运动调控通路的重建。嗅鞘细胞（OECs）移植能促进脊髓损伤后轴突生长和再髓鞘化过程，迄今有关OECs移植对损伤后皮质脊髓束重塑和损伤区神经元再生影响的分子机制尚不清楚。我们前期研究结果显示：Reg-2对脊髓损伤具有神经保护作用。本项目在前期研究基础上，克隆Reg-2基因、构建慢病毒载体并转染OECs，OECs移植治疗脊髓损伤大鼠模型，以期增强OECs促进轴突生长的作用。本研究着重探讨Reg-2基因修饰的OECs对皮质脊髓束轴突再生的影响。采用Reg-2基因克隆、OECs培养、脊髓损伤大鼠模型、神经束路示踪、DTT成像、行为学测试和分子生物学检测等方法，分析OECs对脊髓损伤后皮质脊髓束轴突生长和再髓鞘化、损伤区神经元存活和再生等过程的影响，阐明OECs对脊髓损伤后皮质脊髓束重塑和神经元再生影响的分子机制，为寻找治疗脊髓损伤的新药物和新靶点提供理论依据。

嗅鞘细胞（OECs）移植能促进脊髓损伤后功能恢复。Reg-2对脊髓损伤具有神经保护作用，能促进损伤轴突再生和OECs存活。PLGA支架广泛用于损伤的神经细胞或组织中，用来桥接和促进轴突生长。Reg-2修饰的OECs移植有望增强OECs促进轴突再生作用。本课题构建了脊髓全断损伤SD大鼠模型以及Reg-2过表达质粒，慢病毒包装后转染OECs细胞，进行移植治疗脊髓损伤模型，着重探讨Reg-2基因修饰的OECs对轴突再生的影响。综合采用以下方法：应用差速贴壁法等进行OECs细胞培养纯化，通过PCR扩增、酶切等方法获得含有相应酶切位点的Reg-2基因全长序列。用反转录酶多聚酶链式反应方法和Western Blot测试OECs中重组Reg-2基因的表达。采用TUNEL，BrdU和GFAP等免疫荧光染色分析OECs在体内存活，增殖和迁移分化的情况以及对脊髓神经元存活、轴突再生的影响。在Reg-2转染的OECs移植治疗8周后，比较各组间损伤空洞体积大小、轴突再生、髓鞘形态和动物行为学等差异。采用BBB评分、筛网走道实验和斜板测试对脊髓损伤后运动功能恢复作评估，证实Reg-2转染的OECs移植治疗组与脊髓损伤对照组存在显著差异，OECs移植治疗明显改善脊髓损伤后运动功能状态。免疫组织化学染色借助Map-2、髓鞘碱性蛋白和GFAP免疫组化染色直接显示再生的轴突和胶质斑痕的生长情况。Bilchoski银染、尼氏染色和Fast Blue染色结果提示Reg-2转染的OECs移植治疗组与脊髓损伤对照组相比，OECs促进轴突再生，具有显著差异。TEM观察脊髓损伤后1周脊髓损伤区轴突髓鞘出现髓鞘聚集和脱髓鞘反应。OECs治疗后脊髓损伤区域病变明显减轻，2周后损伤区域出现新的髓鞘和较多的胶质细胞增生。本研究重点分析了 OECs移植对脊髓损伤后皮质脊髓束轴突生长和再髓鞘化，损伤区神经元存活和再生等过程的影响，阐明了Reg-2转染的OECs对损伤后皮质脊髓束重塑和神经元再生以及对脊髓损伤修复作用，为应用基因干预的OECs以及OECs联合PLGA支架治疗脊髓损伤新途径提供重要的理论依据。