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》

作者：

汪雷 ¹ , 宋跃明 ¹ , 袁海峰 ² , 刘立岷 ¹ , 龚全 ¹ , 孔清泉 ¹ , 杨曦 ¹

1 四川大学华西医院骨科（成都，610041）;;
2宁夏医科大学附属总医院脊柱骨科;

关键词：

神经干细胞 NEP1-40 细胞移植脊髓损伤细胞分化大鼠

DOI：

10.7507/1002-1892.20130297

视频：

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摘要 全文 图表 视频 参考文献 施引文献 补充材料

目的探讨NEP1-40（Nogo extracellular peptide residues 1-40）基因修饰对神经干细胞（neural stem cells，NSCs）移植后存活和分化的影响。方法从孕18 d Wistar大鼠胚胎大脑皮质中分离获得原代NSCs并进行体外培养。采用慢病毒载体将NEP1-40基因导入第3代NSCs内完成NEP1-40基因修饰。将30只成年雌性SD大鼠在T9水平进行脊髓右侧半切后随机分为脊髓损伤（spinal cord injury，SCI）组（B组）、NSCs移植组（C组）、NEP1-40基因修饰NSCs组（D组），每组各10只，伤后7 d在损伤局部分别植入NSCs基础培养基、NSCs悬液及NEP1-40基因修饰的NSCs；另取10只SD大鼠仅行T9椎板切除设为假手术组（A组）。移植后8周，取损伤段脊髓组织行辣根过氧化物酶（horseradish peroxidase，HRP）神经示踪、巢蛋白（Nestin）免疫荧光染色及神经丝蛋白200（neurofilament 200，NF-200）、胶质纤维酸性蛋白（glial fibrillary acidic portein，GFAP）、髓鞘碱性蛋白（myelin basic protein，MBP）免疫组织化学染色，评价神经纤维再生情况及移植细胞存活和分化情况。结果移植后8周，A、B、C、D组HRP阳性染色神经纤维束分别为（85.17 ± 6.97）、（12.17 ± 2.79）、（33.58 ± 5.47）、（59.25 ± 7.75）个，B、C、D组显著少于A组（P lt; 0.01）；但C、D组显著高于B组，D组显著高于C组，差异均有统计学意义（P lt; 0.01）。Nestin免疫荧光染色示，A组未见明显Nestin荧光信号，B组仅可见少量散在荧光信号，C、D组有较强荧光信号。免疫组织化学染色定量分析示，NF-200阳性细胞数及MBP积分吸光度（IA）值A组最高，D组次之，C组较少，B组最少，组间两两比较差异均有统计学意义（P lt; 0.05）；GFAP阳性细胞数B组最多，D组次之，C组较少，A组最少，组间两两比较差异均有统计学意义（P lt; 0.05）；C、D组间NF-200、GFAP、MBP阳性细胞百分比比较差异均无统计学意义（P gt; 0.05）。结论 NEP1-40基因修饰对NSCs移植后的分化无明显诱导性，但能促进移植细胞存活、分化及神经元轴突再生，为研究NSCs移植治疗SCI提供了新思路。

引用本文： 汪雷,宋跃明,袁海峰,刘立岷,龚全,孔清泉,杨曦. 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. doi: 10.7507/1002-1892.20130297 复制

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.

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NEP1-40基因修饰对神经干细胞移植后存活和分化的影响

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《中国修复重建外科杂志》

NEP1-40基因修饰对神经干细胞移植后存活和分化的影响

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