NEP1-40基因修飾對神經干細胞移植后存活和分化的影響_《中國修復重建外科雜志》

作者：

汪雷 ¹ , 宋躍明 ¹ , 袁海峰 ² , 劉立岷 ¹ , 龔全 ¹ , 孔清泉 ¹ , 楊曦 ¹

1 四川大學華西醫院骨科（成都，610041）;;
2寧夏醫科大學附屬總醫院脊柱骨科;

關鍵詞：

神經干細胞 NEP1-40 細胞移植脊髓損傷細胞分化大鼠

DOI：

10.7507/1002-1892.20130297

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目的探討NEP1-40（Nogo extracellular peptide residues 1-40）基因修飾對神經干細胞（neural stem cells，NSCs）移植后存活和分化的影響。方法從孕18 d Wistar大鼠胚胎大腦皮質中分離獲得原代NSCs并進行體外培養。采用慢病毒載體將NEP1-40基因導入第3代NSCs內完成NEP1-40基因修飾。將30只成年雌性SD大鼠在T9水平進行脊髓右側半切后隨機分為脊髓損傷（spinal cord injury，SCI）組（B組）、NSCs移植組（C組）、NEP1-40基因修飾NSCs組（D組），每組各10只，傷后7 d在損傷局部分別植入NSCs基礎培養基、NSCs懸液及NEP1-40基因修飾的NSCs；另取10只SD大鼠僅行T9椎板切除設為假手術組（A組）。移植后8周，取損傷段脊髓組織行辣根過氧化物酶（horseradish peroxidase，HRP）神經示蹤、巢蛋白（Nestin）免疫熒光染色及神經絲蛋白200（neurofilament 200，NF-200）、膠質纖維酸性蛋白（glial fibrillary acidic portein，GFAP）、髓鞘堿性蛋白（myelin basic protein，MBP）免疫組織化學染色，評價神經纖維再生情況及移植細胞存活和分化情況。結果移植后8周，A、B、C、D組HRP陽性染色神經纖維束分別為（85.17 ± 6.97）、（12.17 ± 2.79）、（33.58 ± 5.47）、（59.25 ± 7.75）個，B、C、D組顯著少于A組（P lt; 0.01）；但C、D組顯著高于B組，D組顯著高于C組，差異均有統計學意義（P lt; 0.01）。Nestin免疫熒光染色示，A組未見明顯Nestin熒光信號，B組僅可見少量散在熒光信號，C、D組有較強熒光信號。免疫組織化學染色定量分析示，NF-200陽性細胞數及MBP積分吸光度（IA）值A組最高，D組次之，C組較少，B組最少，組間兩兩比較差異均有統計學意義（P lt; 0.05）；GFAP陽性細胞數B組最多，D組次之，C組較少，A組最少，組間兩兩比較差異均有統計學意義（P lt; 0.05）；C、D組間NF-200、GFAP、MBP陽性細胞百分比比較差異均無統計學意義（P gt; 0.05）。結論 NEP1-40基因修飾對NSCs移植后的分化無明顯誘導性，但能促進移植細胞存活、分化及神經元軸突再生，為研究NSCs移植治療SCI提供了新思路。

引用本文： 汪雷,宋躍明,袁海峰,劉立岷,龔全,孔清泉,楊曦. NEP1-40基因修飾對神經干細胞移植后存活和分化的影響. 中國修復重建外科雜志, 2013, 27(11): 1368-1374. doi: 10.7507/1002-1892.20130297 復制

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, 趙紅洋, 溫天蓮, 等. 缺氧缺血性腦損傷新生大鼠腦Nogo受體水平及NEP1-40的神經保護作用. 中華兒科雜志, 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, 宋躍明, 劉浩, 等. NEP1-40 基因慢病毒載體構建及鑒定. 中國修復重建外科雜志, 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.	宮福良, 王坤正, 余鵬博, 等. NEP1-40基因克隆及蛋白的原核表達和純化. 中國修復重建外科雜志, 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.	鄧興力, 梁袁昕, 楊智勇, 等. 神經營養因子3基因修飾神經干細胞移植脊髓損傷的相關蛋白表達. 中國組織工程研究與臨床康復, 2010, 14(36): 6751-6754.
24.	石瑞成, 馮士軍, 王志剛. 高壓氧聯合神經干細胞移植治療大鼠脊髓損傷. 中國組織工程研究與臨床康復, 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, 趙紅洋, 溫天蓮, 等. 缺氧缺血性腦損傷新生大鼠腦Nogo受體水平及NEP1-40的神經保護作用. 中華兒科雜志, 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, 宋躍明, 劉浩, 等. NEP1-40 基因慢病毒載體構建及鑒定. 中國修復重建外科雜志, 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. 宮福良, 王坤正, 余鵬博, 等. NEP1-40基因克隆及蛋白的原核表達和純化. 中國修復重建外科雜志, 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. 鄧興力, 梁袁昕, 楊智勇, 等. 神經營養因子3基因修飾神經干細胞移植脊髓損傷的相關蛋白表達. 中國組織工程研究與臨床康復, 2010, 14(36): 6751-6754.
24. 石瑞成, 馮士軍, 王志剛. 高壓氧聯合神經干細胞移植治療大鼠脊髓損傷. 中國組織工程研究與臨床康復, 2011, 15(19): 3507-3512.

《中國修復重建外科雜志》

NEP1-40基因修飾對神經干細胞移植后存活和分化的影響

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《中國修復重建外科雜志》

NEP1-40基因修飾對神經干細胞移植后存活和分化的影響

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