IL-10转染神经干细胞对脊髓损伤后轴突再生修复的机制研究

After spinal cord injury, the inflammatory cell infiltration and secretion of inflammatory mediators increased tissue injury, while shedding of myelin and glial scar released the myelin associated inhibitory factor that activated of the RhoA pathway and inhibited axon regeneration. In previous study we observed IL-10 as an effective anti-inflammatory factors, can reduce the damage to local inflammation and promote axonal regeneration after injury, but its mechanism is still unknown. With the literature and previous study experiments we hypothesize that IL-10 could regulate local inflammatory response to protect the remnant neurons, on the other hand it could regulat the macrophage phagocytosis to remove the myelin debris and inhibit the RhoA pathway to promote axonal regeneration. The present study intends to construct lentivirus mediated by IL-10 and siRNA respectively transfected to neural stem cells ( NSCs ), up and down-regulation of IL-10 of NSCs, then transplant to cultured neuron injury model and a rat model of spinal cord injury. We will observe the motor function, macrophage phagocytosis, inflammatory response and the changes of RhoA signaling pathway. The result will reveal that mechanism of the IL-10 modified neural stem cells inhibiting of secondary injury, promoting axonal growth and replacing the died neurons to promote neural loop formation function.

脊髓损伤后由于各类炎性细胞侵润并分泌大量炎症介质，加重了组织损伤，同时脱落的髓鞘和胶质瘢痕可释放髓磷脂相关抑制因子激活RhoA通路而抑制轴突再生。前期工作中我们观察到IL-10作为一种有效的抗炎因子，可减轻损伤局部炎症反应并促进轴突损伤后再生修复，但其具体机制未明。结合文献及先前实验我们假设IL-10调节损伤局部炎症反应以保护残存神经元，同时调节巨噬细胞吞噬而迅速清除髓鞘碎片以抑制RhoA通路来促进轴突再生。因此本研究拟构建慢病毒介导的IL-10及其siRNA分别转染神经干细胞（NSCs），通过上、下调IL-10的NSCs移植治疗培养神经元损伤模型和脊髓损伤大鼠模型，研究其对运动功能、损伤局部单核巨噬细胞的吞噬、损伤局部炎症反应及轴突再生抑制RhoA信号通路的影响，其结果可揭示IL-10修饰的神经干细胞在抑制继发性损伤和促进轴突生长和替代死亡神经元促进神经环路形成的作用。

随着经济的发展，交通事故，高处坠落造成的脊髓损伤的发生率呈上升趋势。本研究通过上、下调白介素-10（Interleukin-10，IL-10）的神经干细胞治疗体外神经元氧糖剥夺模型和体内脊髓损伤大鼠模型，对大鼠的运动功能、损伤局部的髓鞘存活情况、损伤局部的炎症反应以及神经元凋亡进行观察，结果发现：在大鼠的运动功能评分中，转染IL-10神经干细胞的大鼠下肢运动恢复较好，表明转染IL-10的NSCs促进神经修复，HE染色示转染IL-10的NSCs组的髓鞘存活较多，Western blot及免疫组化发现转染IL-10的NSCs组的GAP-43阳性细胞增多。在体外神经元氧糖剥夺后与转染IL-10的神经干细胞共培养实验中，TUNEL示转染IL-10的NSCs组的神经元发生凋亡减少。Western Blot检测发现GAP43蛋白水平增加。在进一步的研究中发现IL-10转染神经干细胞通过调控miRNA改变巨噬细胞的表型从而促进轴突生长。通过本研究培养硕士研究生2名，获得成果1项，浙江省医药卫生科技奖二等奖，已经发表国家重点期刊2篇，在投SCI论文1篇。