NEP1-40-overexpressing Neural Stem Cells Enhance Axon Regeneration by Inhibiting Nogo-A/NgR1 Signaling Pathway
NEP1-40-overexpressing Neural Stem Cells Enhance Axon Regeneration by Inhibiting Nogo-A/NgR1 Signaling Pathway
复制标题
NEP1-40 过表达神经干细胞通过抑制 Nogo-A/NgR1 信号通路增强轴突再生
DOI：
10.2174/1567202618666210920115716
复制
发表时间：
2021-01-01
影响因子：
2.1
通讯作者：
Yuan, Haifeng
中科院分区：
文献类型：
--
作者：
Zhang, Bi;Wang, Dalin;Yuan, Haifeng
背景：Nogo-66拮抗肽（NEP1-40）具有改善脊髓损伤（SCI）的潜力。目的：探讨NEP1-40过表达对神经干细胞（NSCs）调控损伤神经元轴突再生的影响。方法：从妊娠大鼠胎儿脑组织中分离NSCs，采用Nestin免疫荧光法对其进行鉴定。通过转染过表达NEP1-40的载体构建过表达的NSCs。通过RT-PCR、western blotting和免疫荧光检测NSCs分化相关标志物Tuj-1、GFAP、Oligo2和MBP的表达。采用NeuN免疫荧光染色法测定神经元数量。western blotting检测LIMK1/2、cofilin、MLC-2磷酸化水平及GAP-43、MAP-2、APP蛋白表达水平。结果：NEP1-40过表达可促进Tuj-1、Oligo2、MBP蛋白表达水平，增加Tuj-1、Oligo2、MBP阳性细胞数量。过表达nep1 -40的NSCs （NEP-NSCs）改善了与损伤神经元共培养的NeuN阳性细胞。NEP-NSCs还增加了轴突再生指标（GAP-43、MAP-2）的蛋白水平，降低了APP蛋白水平。此外，NEP-NSCs中LIMK1/2、cofilin和MLC-2的磷酸化水平明显降低。结论：NEP1-40过表达通过抑制Nogo-A/NgR1信号通路，增强NSCs向神经元分化的能力，促进轴突再生。本研究为脊髓损伤的治疗提供了一种新的基因修饰移植方法。
Background: Nogo-66 antagonistic peptide (NEP1-40) offers the potential to improve spinal cord injury (SCI). Objective: To explore the effect of NEP1-40 overexpression on neural stem cells (NSCs) regulating the axon regeneration of injured neurons. Methods: We isolated NSCs from brain tissues of pregnant rat fetuses and used Nestin immunofluorescence to identify them. The NEP1-40 overexpressing NSCs were constructed by transfection with the NEP1-40-overexpressing vector. The expression of NSCs differentiation associated markers, including Tuj-1, GFAP, Oligo2, and MBP, were detected by RT-PCR, western blotting, and immunofluorescence. NeuN immunofluorescence staining was used to measure the number of neurons. And western blotting was used to detect the phosphorylation levels of LIMK1/2, cofilin, and MLC-2 and the protein levels of GAP-43, MAP-2, and APP. Results: The NEP1-40 overexpression promoted the expression level of Tuj-1, Oligo2, and MBP, and increased the number of Tuj-1, Oligo2, and MBP positive cells. NEP1-40-overexpressing NSCs (NEP-NSCs) improved NeuN positive cells of co-culture with injured neurons. And NEP-NSCs also increased the protein levels of axon regeneration indicators (GAP-43, MAP-2) and decreased APP protein level. In addition, the phosphorylation level of LIMK1/2, cofilin, and MLC-2 were markedly decreased in NEP-NSCs. Conclusion: NEP1-40 overexpression enhanced the ability of NSCs differentiation into neurons and promoted axon regeneration by inhibiting the Nogo-A/NgR1 signaling pathway. This study provides an alternative gene modified transplantation NSCs for the SCI treatment.