中国修复重建外科杂志

中国修复重建外科杂志

慢病毒介导 NEP1-40 及 NT-3 双基因转染神经干细胞的实验研究

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目的通过慢病毒载体将 NEP1-40(Nogo extracellular peptide residues 1-40)及神经营养因子 3(neurotrophin 3,NT-3)双基因转染入神经干细胞(neural stem cells,NSCs)内进行表达,探讨 NEP1-40 及 NT-3 双基因转染 NSCs 的可行性,为 NSCs 体内实验奠定基础。方法将 SD 大鼠胚胎室管膜区 NSCs 采用空载慢病毒载体(A 组)、NEP1-40 慢病毒载体(B 组)、NT-3 慢病毒载体(C 组)及 NEP1-40 和 NT-3 慢病毒载体(D 组)进行转染,以未转染病毒的细胞作为对照组(E 组)。用感染复数(multiplicity of infection,MOI)为 5、10、15 的慢病毒载体分别转染 24、48、72 h,荧光显微镜观察转染后细胞内荧光表达情况,确定慢病毒载体的最佳 MOI 和收样时间。再分别通过实时荧光定量 PCR 及 Western blot,检测转染后细胞中 NEP1-40 及 NT-3 基因的表达,以及细胞和培养基中 NEP1-40 及 NT-3 蛋白的表达。结果荧光显微镜观察示,MOI 为 10 时 NEP1-40 和 NT-3 基因慢病毒载体在 NSCs 内转染率最高,最佳时间为转染 48 h 时。实时荧光定量 PCR 及 Western blot 检测示,B、D 组 NEP1-40 mRNA 相对表达量和蛋白相对表达量均显著高于 A、C 组,差异有统计学意义(P<0.05);A、C 组间及 B、D 组间比较差异无统计学意义(P>0.05)。C、D 组 NT-3 mRNA 相对表达量和蛋白相对表达量均显著高于 A、B 组,差异有统计学意义(P<0.05);A、B 组间和 C、D 组间比较差异无统计学意义(P>0.05)。结论通过慢病毒载体可将 NEP1-40 及 NT-3 双基因成功转染入 NSCs 内,在 NSCs 内稳定表达,且两种目的基因在表达过程中无相互拮抗或促进作用。

ObjectiveTo explore the feasibility of co-transduction and co-expression of Nogo extracellular peptide residues 1-40 (NEP1-40) gene and neurotrophin 3 (NT-3) gene into neural stem cells (NSCs).MethodsNSCs were derived from the cortex tissue of Sprague Dawley rat embryo. The experiment included 5 groups: no-load lentiviral vector transducted NSCs (group A), NEP1-40 transducted NSCs (group B), NT-3 transducted NSCs (group C), NEP1-40 and NT-3 corporately transducted NSCs (group D), and blank control (group E). Target genes were transducted into NSCs by lentiviral vectors of different multiplicity of infection (MOI; 5, 10, 15) for different time (24, 48, 72 hours). Fluorescent microscope was used to observe the expression of fluorescence protein and acquire the optimum MOI and optimum collection time. Real-time fluorescence quantitative PCR and Western blot tests were utilized to evaluate the gene expressions of NEP1-40 and NT-3 in NSCs and protein expressions of NEP1-40 and NT-3 in NSCs and in culture medium.ResultsThe optimum MOI for both target gene was 10 and the optimum collection time was 48 hours. The real-time fluorescence quantitative PCR and Western blot results showed that the mRNA and protein relative expressions of NEP1-40 in groups B and D were significantly higher than those in groups A and C (P<0.05), but no significant difference was found between groups B and D, and between groups A and C (P>0.05). The mRNA and protein relative expressions of NT-3 in groups C and D were significantly higher than those in groups A and B (P<0.05), but no significant difference was found between groups A and B, and between groups C and D (P>0.05).ConclusionNEP1-40 and NT-3 gene can be successfully co-transducted into NSCs by the mediation of lentiviral vector. The expressions of the two target genes are stable and have no auxo-action or antagonism between each other.

关键词: 神经干细胞; NEP1-40; 神经营养因子 3; 慢病毒载体; 转染

Key words: Neural stem cells; Nogo extracellular peptide residues 1-40; neurotrophin 3; lentiviral vector; transduction

引用本文: 王林楠, 汪雷, 宋跃明, 刘立岷, 杨曦, 丰干均, 周春光. 慢病毒介导 NEP1-40 及 NT-3 双基因转染神经干细胞的实验研究. 中国修复重建外科杂志, 2018, 32(4): 420-427. doi: 10.7507/1002-1892.201710079 复制

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1. Kabatas S, Teng YD. Potential roles of the neural stem cell in the restoration of the injured spinal cord: review of the literature. Turk Neurosurg, 2010, 20(2): 103-110.
2. Cai PQ, Sun GY, Cai PS, et al. Survival of transplanted neurotrophin-3 expressing human neural stem cells and motor function in a rat model of spinal cord injury. Neural Regen Res, 2009, 4(7): 485-491.
3. Salewski RP, Mitchell RA, Shen C, et al. Transplantation of neural stem cells clonally derived from embryonic stem cells promotes recovery after murine spinal cord injury. Stem Cells Dev, 2015, 24(1): 36-50.
4. Steward O, Sharp K, Yee KM, et al. A re-assessment of the effects of a Nogo-66 receptor antagonist on regenerative growth of axons and locomotor recovery after spinal cord injury in mice. Exp Neurol, 2008, 209(2): 446-468.
5. Zahir T, Chen YF, MacDonald JF, et al. Neural stem/progenitor cells differentiate in vitro to neurons by the combined action of dibutyryl cAMP and interferon-gamma. Stem Cells Dev, 2009, 18(10): 1423-1432.
6. Atalay B, Bavbek M, Ozen O, et al. Nogo-A inhibitory peptide (NEP1-40) increases pan-cadherin expression following mild cortical contusion injury in rats. Turk Neurosurg, 2008, 18(4): 356-365.
7. Duricki DA, Huston TH, Kathe C, et al. Delayed intramuscular human neurotrophin-3improves recovery in adult and elderly rats after stroke. Brain, 2016, 139(Pt 1): 259-275.
8. Lu HX, Hao ZM, Jiao Q, et al. Neurotrophin-3 gene transduction of mouse neural stem cells promotes proliferation and neuronal differentiation in organotypic hippocampal slice cultures. Med Sci Monit, 2011, 17(11): 305-311.
9. 汪雷, 宋跃明, 刘立岷, 等. NEP1-40 基因修饰的神经干细胞移植对脊髓损伤大鼠行为学恢复的影响. 华西医学, 2014, 29(11): 2006-2011.
10. Corti S, Locatelli F, Papadimitriou D, et al. Somatic stem cell research for neural repair: current evidence and emerging perspectives. J Cell Mol Med, 2004, 8(3): 329-337.
11. Hou T, Shi Y, Cheng SG, et al. Nogo-A expresses on neural stem cell surface. Int J Neurosci, 2010, 120(3): 201-205.
12. Steward O, Sharp K, Yee KM, et al. A re-assessment of the effects of a Nogo-66 receptor antagonist on regenerative growth of axons and locomotor recovery after spinal cord injury in mice. Exp Neurol, 2008, 209(2): 446-468.
13. Dupuis L, Pehar M, Cassina P, et al. Nogo receptor antagonizes p75NTR-dependent motor neuron death. Proc Natl Acad Sci U S A, 2008, 105(2): 740-745.
14. Atalay B, Bavbek M, Cekinmez M, et al. Antibodies neutralizing Nogo-A increase pan-cadherin expression and motor recovery following spinal cord injury in rats. Spinal Cord, 2007, 45(12): 780-786.
15. Gou X, Wang Q, Yang Q, et al. TAT-NEP1-40 as a novel therapeutic candidate for axonal regeneration and functional recovery after stroke. J Drug Target, 2010, 19(2): 86-95.
16. 袁海峰, 杨先森, 胡利红, 等. 慢病毒介导 NEP1-40 转染神经干细胞的研究. 宁夏医科大学学报, 2015, 37(12): 1375-1378.
17. 汪雷, 宋跃明, 袁海峰, 等. NEP1-40 基因修饰对神经干细胞移植后存活和分化的影响. 中国修复重建外科杂志, 2013, 27(11): 1368-1374.
18. Boyce VS, Mendell LM. Neurotrophic factors in spinal cord injury. Handb Exp Pharmacol, 2014, 220: 443-460.
19. Abu El-Asrar AM, Mohammad G, De Hertogh G, et al. Neurotrophins and neurotrophin receptors in proliferative diabetic retinopathy. PLoS One, 2013, 8(6): e654-672.
20. Zhang YQ, He LM, Xing B, et al. Neurotrophin-3 gene-modified Schwann cells promote TrkC gene-modified mesenchymal stem cells to differentiate into neuron-like cells in poly (lactic-acid-co-glycolic acid) multiple-channel conduit. Cells Tissues Organs, 2011, 195(4): 313-322.
21. Bambakidis NC, Miller RH. Transplantation of oligodendrocyte precursors and sonic hedgehog results in improved function and white matter sparing in the spinal cords of adult rats after contusion. Spine J, 2004, 4(1): 16-26.
22. 徐委, 程黎明. 神经营养因子修复脊髓损伤的研究与应用. 中国组织工程研究, 2013, 17(2): 369-374.
23. Zhang L, Gu S, Zhao C, et al. Combined treatment of neurotrophin-3 gene and neural stem cells is propitious to functional recovery after spinal cord injury. Cell Transplant, 2007, 16(5): 475-481.