Experimental study of lentivirus-mediated Nogo extracellular peptide residues 1-40 gene and neurotrophin 3 gene co-transduction in neural stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Linnan ,  WANG Lei , SONG Yueming , LIU Limin , YANG Xi , FENG Ganjun , ZHOU Chunguang

Department of Orthopedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China;

Corresponding author：

WANG Lei, Email: wangle_cd@126.com

Keywords：

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

DOI：

10.7507/1002-1892.201710079

Video：

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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.

Citation： WANG Linnan, WANG Lei, SONG Yueming, LIU Limin, YANG Xi, FENG Ganjun, ZHOU Chunguang. Experimental study of lentivirus-mediated Nogo extracellular peptide residues 1-40 gene and neurotrophin 3 gene co-transduction in neural stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(4): 420-427. doi: 10.7507/1002-1892.201710079 Copy

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.

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.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study of lentivirus-mediated Nogo extracellular peptide residues 1-40 gene and neurotrophin 3 gene co-transduction in neural stem cells

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