人膠質細胞源性神經營養因子基因修飾猴雪旺細胞的構建與鑒定_《中國修復重建外科雜志》

作者：

巴越洋 , 王輝 , 寧昕杰

中山大學附屬第三醫院神經外科（廣州，510630）;

關鍵詞：

人膠質源性神經營養因子重組逆轉錄病毒基因轉染雪旺細胞恒河猴

DOI：

10.7507/1002-1892.20130296

視頻：

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目的構建人膠質細胞源性神經營養因子（human glial cell derived neurotrophic factor，hGDNF）基因修飾的猴雪旺細胞穩定細胞系。方法以pUC19-hGDNF為模板，擴增含hGDNF基因序列，將其插入質粒pBABE-puro，構建pBABE-puro-hGDNF重組真核表達載體，經酶切鑒定及DNA測序鑒定重組質粒。從恒河猴提取、培養、純化雪旺細胞。包裝病毒，利用含hGDNF基因的重組逆轉錄病毒轉染雪旺細胞，實時熒光定量PCR、Western blot檢測hGDNF mRNA及蛋白表達情況。結果PCR產物hGDNF基因編碼序列大小約596 bp。重組表達載體經雙酶切鑒定，含有1 條596 bp的特異性條帶，其基因測序結果與基因庫中hGDNF基因序列片段相同，提示hGDNF基因成功轉入逆轉錄病毒。逆轉錄病毒轉染的雪旺細胞hGDNF mRNA及蛋白表達量顯著高于未轉染雪旺細胞，差異有統計學意義（P lt; 0.05）。結論成功構建hGDNF基因修飾的猴雪旺細胞穩定細胞系，能長期穩定高效釋放hGDNF，為進一步將其應用于神經損傷修復奠定了基礎。

引用本文： 巴越洋,王輝,寧昕杰. 人膠質細胞源性神經營養因子基因修飾猴雪旺細胞的構建與鑒定. 中國修復重建外科雜志, 2013, 27(11): 1363-1367. doi: 10.7507/1002-1892.20130296 復制

1.	Johnson EO, Zoubos AB, Soucacos PN. Regeneration and repair of peripheral nerves. Injury, 2005, 36 Suppl 4: S24-S29.
2.	Wood MD, Gordon T, Kim H, et al. Fibrin gels containing GDNF microspheres increase axonal regeneration after delayed peripheral nerve repair. Regen Med, 2013, 8(1): 27-37.
3.	Guzen FP, de Almeida Leme RJ, de Andrade MS, et al. Glial cell line-derived neurotrophic factor added to a sciatic nerve fragment grafted in a spinal cord gap ameliorates motor impairments in rats and increases local axonal growth. Restor Neurol Neurosci, 2009, 27(1): 1-16.
4.	Kokai LE, Ghaznavi AM, Marra KG. Incorporation of double-walled microspheres into polymer nerve guides for the sustained delivery of glial cell line-derived neurotrophic factor. Biomaterials, 2010, 31(8): 2313-2322.
5.	Kokai LE, Bourbeau D, Weber D, et al. Sustained growth factor delivery promotes axonal regeneration in long gap peripheral nerve repair. Tissue Eng Part A, 2011, 17(9-10): 1263-1275.
6.	Mills CD, Allchorne AJ, Griffin RS, et al. GDNF selectively promotes regeneration of injury-primed sensory neurons in the lesioned spinal cord. Mol Cell Neurosci, 2007, 36(2): 185-194.
7.	Tian H, Wu JY, Shang YJ, et al. Expression and immunological analysis of capsid protein precursor of swine vesicular disease virus HK/70. Virol Sin, 2010, 25(3): 206-212.
8.	Gordon T. The role of neurotrophic factors in nerve regeneration. Neurosurg Focus, 2009, 26(2): E3.
9.	Lin LF, Doherty DH, Lile JD, et al. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science, 1993, 260(5111): 1130-1132.
10.	Wood MD, Gordon T, Kemp SW, et al. Functional motor recovery is improved due to local placement of gdnf microspheres after delayed nerve repair. Biotechnol Bioeng, 2013, 110(5): 1272-1281.
11.	Wood MD, Kim H, Bilbily A, et al. GDNF released from microspheres enhances nerve regeneration after delayed repair. Muscle Nerve, 2012, 46(1): 122-124.
12.	Madduri S, di Summa P, Papaloizos M, et al. Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats. Biomaterials, 2010, 31(32): 8402-8409.
13.	Sugiura Y, Lin W. Neuron-glia interactions: the roles of schwann cells in neuromuscular synapse formation and function. Biosci Rep, 2011, 31(5): 295-302.
14.	Barras FM, Kuntzer T, Zurn AD, et al. Local delivery of glial cell line-derived neurotrophic factor improves facial nerve regeneration after late repair. Laryngoscope, 2009, 119(5): 846-855.
15.	May F, Buchner A, Schlenker B, et al. Schwann cell-mediated delivery of glial cell line-derived neurotrophic factor restores erectile function after cavernous nerve injury. Int J Urol, 2013, 20(3): 344-348.
16.	May F, Matiasek K, Vroemen M, et al. GDNF-transduced Schwann cell grafts enhance regeneration of erectile nerves. Eur Urol, 2008, 54(5): 1179-1187.
17.	Hood B, Levene HB, Levi AD. Transplantation of autologous Schwann cells for the repair of segmental peripheral nerve defects. Neurosurg Focus, 2009, 26(2): E4.
18.	Gravvanis AI, Lavdas AA, Papalois A, et al. The beneficial effect of genetically engineered schwann cells with enhanced motility in peripheral nerve regeneration: review. Acta Neurochir Suppl, 2007, 100: 51-56.
19.	Vannucci L, Lai M, Chiuppesi F, et al. Viral vectors: a look back and ahead on gene transfer technology. New Microbiol, 2013, 36(1): 1-22.
20.	Chtarto A, Yang X, Bockstael O, et al. Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector. Exp Neurol, 2007, 204(1): 387-399.
21.	Yoo YM, Lee CJ, Lee U, et al. Neuroprotection of adenoviral-vector-mediated GDNFexpression against kainic-acid-induced excitotoxicity in the rat hippocampus. Exp Neurol, 2006, 200(2): 407-417.
22.	Araki K, Shiotani A, Watabe K, et al. Adenoviral GDNF gene transfer enhances neurofunctional recovery after recurrent laryngeal nerve injury. Gene Ther, 2006, 13(4): 296-303.
23.	張文捷, 李兵倉, 任先軍, 等. pLXSN-GDNF重組載體的鑒定及其包裝細胞系的建立. 中國修復重建外科雜志, 2006, 20(11): 1119-1123.

1. Johnson EO, Zoubos AB, Soucacos PN. Regeneration and repair of peripheral nerves. Injury, 2005, 36 Suppl 4: S24-S29.
2. Wood MD, Gordon T, Kim H, et al. Fibrin gels containing GDNF microspheres increase axonal regeneration after delayed peripheral nerve repair. Regen Med, 2013, 8(1): 27-37.
3. Guzen FP, de Almeida Leme RJ, de Andrade MS, et al. Glial cell line-derived neurotrophic factor added to a sciatic nerve fragment grafted in a spinal cord gap ameliorates motor impairments in rats and increases local axonal growth. Restor Neurol Neurosci, 2009, 27(1): 1-16.
4. Kokai LE, Ghaznavi AM, Marra KG. Incorporation of double-walled microspheres into polymer nerve guides for the sustained delivery of glial cell line-derived neurotrophic factor. Biomaterials, 2010, 31(8): 2313-2322.
5. Kokai LE, Bourbeau D, Weber D, et al. Sustained growth factor delivery promotes axonal regeneration in long gap peripheral nerve repair. Tissue Eng Part A, 2011, 17(9-10): 1263-1275.
6. Mills CD, Allchorne AJ, Griffin RS, et al. GDNF selectively promotes regeneration of injury-primed sensory neurons in the lesioned spinal cord. Mol Cell Neurosci, 2007, 36(2): 185-194.
7. Tian H, Wu JY, Shang YJ, et al. Expression and immunological analysis of capsid protein precursor of swine vesicular disease virus HK/70. Virol Sin, 2010, 25(3): 206-212.
8. Gordon T. The role of neurotrophic factors in nerve regeneration. Neurosurg Focus, 2009, 26(2): E3.
9. Lin LF, Doherty DH, Lile JD, et al. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science, 1993, 260(5111): 1130-1132.
10. Wood MD, Gordon T, Kemp SW, et al. Functional motor recovery is improved due to local placement of gdnf microspheres after delayed nerve repair. Biotechnol Bioeng, 2013, 110(5): 1272-1281.
11. Wood MD, Kim H, Bilbily A, et al. GDNF released from microspheres enhances nerve regeneration after delayed repair. Muscle Nerve, 2012, 46(1): 122-124.
12. Madduri S, di Summa P, Papaloizos M, et al. Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats. Biomaterials, 2010, 31(32): 8402-8409.
13. Sugiura Y, Lin W. Neuron-glia interactions: the roles of schwann cells in neuromuscular synapse formation and function. Biosci Rep, 2011, 31(5): 295-302.
14. Barras FM, Kuntzer T, Zurn AD, et al. Local delivery of glial cell line-derived neurotrophic factor improves facial nerve regeneration after late repair. Laryngoscope, 2009, 119(5): 846-855.
15. May F, Buchner A, Schlenker B, et al. Schwann cell-mediated delivery of glial cell line-derived neurotrophic factor restores erectile function after cavernous nerve injury. Int J Urol, 2013, 20(3): 344-348.
16. May F, Matiasek K, Vroemen M, et al. GDNF-transduced Schwann cell grafts enhance regeneration of erectile nerves. Eur Urol, 2008, 54(5): 1179-1187.
17. Hood B, Levene HB, Levi AD. Transplantation of autologous Schwann cells for the repair of segmental peripheral nerve defects. Neurosurg Focus, 2009, 26(2): E4.
18. Gravvanis AI, Lavdas AA, Papalois A, et al. The beneficial effect of genetically engineered schwann cells with enhanced motility in peripheral nerve regeneration: review. Acta Neurochir Suppl, 2007, 100: 51-56.
19. Vannucci L, Lai M, Chiuppesi F, et al. Viral vectors: a look back and ahead on gene transfer technology. New Microbiol, 2013, 36(1): 1-22.
20. Chtarto A, Yang X, Bockstael O, et al. Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector. Exp Neurol, 2007, 204(1): 387-399.
21. Yoo YM, Lee CJ, Lee U, et al. Neuroprotection of adenoviral-vector-mediated GDNFexpression against kainic-acid-induced excitotoxicity in the rat hippocampus. Exp Neurol, 2006, 200(2): 407-417.
22. Araki K, Shiotani A, Watabe K, et al. Adenoviral GDNF gene transfer enhances neurofunctional recovery after recurrent laryngeal nerve injury. Gene Ther, 2006, 13(4): 296-303.
23. 張文捷, 李兵倉, 任先軍, 等. pLXSN-GDNF重組載體的鑒定及其包裝細胞系的建立. 中國修復重建外科雜志, 2006, 20(11): 1119-1123.

《中國修復重建外科雜志》

人膠質細胞源性神經營養因子基因修飾猴雪旺細胞的構建與鑒定

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《中國修復重建外科雜志》

人膠質細胞源性神經營養因子基因修飾猴雪旺細胞的構建與鑒定

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