CONSTRUCTION AND IDENTIFICATION OF RHESUS MONKEY Schwann CELLS MODIFIED WITH HUMAN GLIAL CELL DERIVED NEUROTROPHIC FACTOR GENE_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

BA Yueyang , WANG Hui , NING Xinjie.

Department of Neurosurgery, the Third Affiliated Hospital, Sun Yat-sen University, Guangzhou Guangdong, 510630, P.R.China. Corresponding author: WANG Hui, E-mail: doctorwanghui@126.com;

Corresponding author：

Keywords：

Glial cell derived neurotrophic factor; Recombinant retrovirus; Gene transfection; Schwann cells; Rhesus monkey

DOI：

10.7507/1002-1892.20130296

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Objective To construct the rhesus monkey Schwann cells (SCs) modified with human glial cell derived neurotrophic factor (hGDNF) gene. Methods The coding sequence of hGDNF amplified by PCR from pUC19-hGDNF was inserted into eukaryotic expression vector pBABE-puro. The recombinant eukaryotic expression vector pBABE-puro-hGDNF was identified with restriction enzyme digestion and DNA sequencing. The SCs were isolated from rhesus monkeys, cultured and purified. The SCs were transfected with the recombinant retrovirus vector containing hGDNF gene. The mRNA and protein expressions of hGDNF were analyzed by real-time fluorescent quantitative PCR and Western blot. Results The PCR product of hGDNF coding sequence was a 596 bp specific segment. The recombinant eukaryotic expression vector was digested into a 596 bp specific segment by specific restriction enzyme and another segment. The 596 bp segment confirmed by DNA sequencing was consistent with hGDNF sequence on GenBank. Restriction enzyme digestion and sequencing results showed that the coding sequence of hGDNF was successfully inserted into the recombinant retrovirus vector and the mRNA and protein expressions of hGDNF were significantly higher in transfected SCs than non-transfected SCs (P lt; 0.05). Conclusion The rhesus monkey SCs modified with hGDNF gene are successfully constructed and hGDNF can be released continuously and stably, which will provide a foundation for the further research about cell therapy of hGDNF-SCs in the repair of injured nerve.

Citation： BA Yueyang,WANG Hui,NING Xinjie.. CONSTRUCTION AND IDENTIFICATION OF RHESUS MONKEY Schwann CELLS MODIFIED WITH HUMAN GLIAL CELL DERIVED NEUROTROPHIC FACTOR GENE. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(11): 1363-1367. doi: 10.7507/1002-1892.20130296 Copy

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.	张文捷, 李兵仓, 任先军, 等. pLXSN-GDNF重组载体的鉴定及其包装细胞系的建立. 中国修复重建外科杂志, 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. 张文捷, 李兵仓, 任先军, 等. pLXSN-GDNF重组载体的鉴定及其包装细胞系的建立. 中国修复重建外科杂志, 2006, 20(11): 1119-1123.

Chinese Journal of Reparative and Reconstructive Surgery

CONSTRUCTION AND IDENTIFICATION OF RHESUS MONKEY Schwann CELLS MODIFIED WITH HUMAN GLIAL CELL DERIVED NEUROTROPHIC FACTOR GENE

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