INFLUENCE OF Nogo EXTRACELLULAR PEPTIDE RESIDUES 1-40 GENE MODIFICATION ON SURVIVAL AND DIFFERENTIATION OF NEURAL STEM CELLS AFTER TRANSPLANTATION_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Lei ¹ , SONG Yueming ¹ , YUAN Haifeng ² , LIU Limin ¹ , GONG Quan ¹ , KONG Qingquan ¹ , YANG Xi ¹

1Department of Orthopedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China;;
2Department of Spinal Orthopedics, Affiliated General Hospital, Ningxia Medical University. Corresponding author: SONG Yueming, E-mail: sym_cd@yahoo.com.cn;

Corresponding author：

Keywords：

Neural stem cells; Nogo extra cellular peptide residues 1-40; Cell transplantation; Spinal cord injury; Differentiation; Rat

DOI：

10.7507/1002-1892.20130297

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Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.

Citation： WANG Lei,SONG Yueming,YUAN Haifeng,LIU Limin,GONG Quan,KONG Qingquan,YANG Xi. INFLUENCE OF Nogo EXTRACELLULAR PEPTIDE RESIDUES 1-40 GENE MODIFICATION ON SURVIVAL AND DIFFERENTIATION OF NEURAL STEM CELLS AFTER TRANSPLANTATION. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(11): 1368-1374. doi: 10.7507/1002-1892.20130297 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.	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.
3.	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.
4.	朱薇薇, 赵红洋, 温天莲, 等. 缺氧缺血性脑损伤新生大鼠脑Nogo受体水平及NEP1-40的神经保护作用. 中华儿科杂志, 2010, 48 (2): 138-142.
5.	Wang Q, Gou X, Xiong L, et al. Trans-activator of transcription-mediated delivery of NEP1-40 protein into brain has a neuroprotective effect against focal cerebral ischemic injury via inhibition of neuronal apoptosis. Anesthesiology, 2008, 108(6): 1071-1080.
6.	袁海峰, 宋跃明, 刘浩, 等. NEP1-40 基因慢病毒载体构建及鉴定. 中国修复重建外科杂志, 2012, 26(2): 177-181.
7.	Li Y, Zhang WM, Wang TH. Optimal location and time for neural stem cell transplantation into transected rat spinal cord. Cell Mol Neurobiol, 2011, 31(3): 407-414.
8.	Mcmahon SS, Albermann S, Rooney GE, et al. Engraftment, migration and differentiation of neural stem cells in the rat spinal cord following contusion injury. Cytotherapy, 2010, 12(3): 313-325.
9.	Maio S, Chen H, Schwab ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an monoclonal antibody IN-1. Nature, 2000, 403(6768): 434-439.
10.	Williams G, Wood A, Williams EJ, et al. Ganglioside inhibition of neurite outgrowth requires Nogo receptor function: identification of interaction sites and development of novel antagonists. J Biol Chem, 2008, 283(24): 16641-16652.
11.	Su Z, Cao L, Zhu Y, et al. Nogo enhances the adhesion of olfactory ensheathing cells and inhibits their migration. J Cell Sci, 2007, 120(Pt 11): 1877-1887.
12.	Hou T, Shi Y, Cheng SG, et al. Nogo-A expresses on neural stem cell Surface. Int J Neurosci, 2010, 120(3): 201-205.
13.	GrandPré T, Li S, Strittmatter SM. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature, 2002, 417(6888): 547-551.
14.	Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci, 2003, 23(10): 4219-4227.
15.	Bart J, Groen HJ, Hendrikse NH, et al. The blood-brain barrier and oncology: new insights into function and modulation. Cancer Treat Rev, 2000, 26(6): 449-462.
16.	宫福良, 王坤正, 余鹏博, 等. NEP1-40基因克隆及蛋白的原核表达和纯化. 中国修复重建外科杂志, 2006, 20(1): 9-12.
17.	Kirik OV, Korzhevskii DE. Expression of neural stem cell marker nestin in the kidney of rats and humans. Bull Exp Biol Med, 2009, 147(4): 539-541.
18.	Andressen C, Stocker E, Klinz FJ, et al. Nestin-specfic green fluo-rescent expression in embryonic stem cell-derived neural precursor cells used for transplantation. Stem Cells, 2001, 19(5): 419-424.
19.	Perrot R, Berges R, Bocquet A, et al. Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Mol Neurobiol, 2008, 38(1): 27-65.
20.	Naghdi M, Tiraihi T, Mesbah-Namin SA, et al. Induction of bone marrow stromal cells into cholinergic-like cells by nerve growth factor. Iran Biomed J, 2009, 13(2): 117-123.
21.	Cayre M, Bancila M, Virard I, et al. Migrating and myelinating potential of subventricular zone neural progenitor cells in white matter tracts of the adult rodent brain. Mol Cell Neurosci, 2006, 31(4): 748-758.
22.	Doetsch F, Caille I, Lim DA, et al. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell, 1999, 97(6): 703-716.
23.	邓兴力, 梁袁昕, 杨智勇, 等. 神经营养因子3基因修饰神经干细胞移植脊髓损伤的相关蛋白表达. 中国组织工程研究与临床康复, 2010, 14(36): 6751-6754.
24.	石瑞成, 冯士军, 王志刚. 高压氧联合神经干细胞移植治疗大鼠脊髓损伤. 中国组织工程研究与临床康复, 2011, 15(19): 3507-3512.

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. 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.
3. 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.
4. 朱薇薇, 赵红洋, 温天莲, 等. 缺氧缺血性脑损伤新生大鼠脑Nogo受体水平及NEP1-40的神经保护作用. 中华儿科杂志, 2010, 48 (2): 138-142.
5. Wang Q, Gou X, Xiong L, et al. Trans-activator of transcription-mediated delivery of NEP1-40 protein into brain has a neuroprotective effect against focal cerebral ischemic injury via inhibition of neuronal apoptosis. Anesthesiology, 2008, 108(6): 1071-1080.
6. 袁海峰, 宋跃明, 刘浩, 等. NEP1-40 基因慢病毒载体构建及鉴定. 中国修复重建外科杂志, 2012, 26(2): 177-181.
7. Li Y, Zhang WM, Wang TH. Optimal location and time for neural stem cell transplantation into transected rat spinal cord. Cell Mol Neurobiol, 2011, 31(3): 407-414.
8. Mcmahon SS, Albermann S, Rooney GE, et al. Engraftment, migration and differentiation of neural stem cells in the rat spinal cord following contusion injury. Cytotherapy, 2010, 12(3): 313-325.
9. Maio S, Chen H, Schwab ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an monoclonal antibody IN-1. Nature, 2000, 403(6768): 434-439.
10. Williams G, Wood A, Williams EJ, et al. Ganglioside inhibition of neurite outgrowth requires Nogo receptor function: identification of interaction sites and development of novel antagonists. J Biol Chem, 2008, 283(24): 16641-16652.
11. Su Z, Cao L, Zhu Y, et al. Nogo enhances the adhesion of olfactory ensheathing cells and inhibits their migration. J Cell Sci, 2007, 120(Pt 11): 1877-1887.
12. Hou T, Shi Y, Cheng SG, et al. Nogo-A expresses on neural stem cell Surface. Int J Neurosci, 2010, 120(3): 201-205.
13. GrandPré T, Li S, Strittmatter SM. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature, 2002, 417(6888): 547-551.
14. Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci, 2003, 23(10): 4219-4227.
15. Bart J, Groen HJ, Hendrikse NH, et al. The blood-brain barrier and oncology: new insights into function and modulation. Cancer Treat Rev, 2000, 26(6): 449-462.
16. 宫福良, 王坤正, 余鹏博, 等. NEP1-40基因克隆及蛋白的原核表达和纯化. 中国修复重建外科杂志, 2006, 20(1): 9-12.
17. Kirik OV, Korzhevskii DE. Expression of neural stem cell marker nestin in the kidney of rats and humans. Bull Exp Biol Med, 2009, 147(4): 539-541.
18. Andressen C, Stocker E, Klinz FJ, et al. Nestin-specfic green fluo-rescent expression in embryonic stem cell-derived neural precursor cells used for transplantation. Stem Cells, 2001, 19(5): 419-424.
19. Perrot R, Berges R, Bocquet A, et al. Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Mol Neurobiol, 2008, 38(1): 27-65.
20. Naghdi M, Tiraihi T, Mesbah-Namin SA, et al. Induction of bone marrow stromal cells into cholinergic-like cells by nerve growth factor. Iran Biomed J, 2009, 13(2): 117-123.
21. Cayre M, Bancila M, Virard I, et al. Migrating and myelinating potential of subventricular zone neural progenitor cells in white matter tracts of the adult rodent brain. Mol Cell Neurosci, 2006, 31(4): 748-758.
22. Doetsch F, Caille I, Lim DA, et al. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell, 1999, 97(6): 703-716.
23. 邓兴力, 梁袁昕, 杨智勇, 等. 神经营养因子3基因修饰神经干细胞移植脊髓损伤的相关蛋白表达. 中国组织工程研究与临床康复, 2010, 14(36): 6751-6754.
24. 石瑞成, 冯士军, 王志刚. 高压氧联合神经干细胞移植治疗大鼠脊髓损伤. 中国组织工程研究与临床康复, 2011, 15(19): 3507-3512.

Chinese Journal of Reparative and Reconstructive Surgery

INFLUENCE OF Nogo EXTRACELLULAR PEPTIDE RESIDUES 1-40 GENE MODIFICATION ON SURVIVAL AND DIFFERENTIATION OF NEURAL STEM CELLS AFTER TRANSPLANTATION

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