EFFECT OF RECOMBINANT CO-EXPRESSION ADENOVIRUS OF NERVE GROWTH FACTOR AND MYELIN ASSOCIATED GLYCOPROTEIN GENES ON RAT SCIATIC NERVE INJURY_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHENYu ¹ , DENGZhongliang ² , WENGZheng ¹ , CHENShimou ¹ ,  TONGWei ¹

1. The Fisrt Department of Orthopedics, Chongqing Ninth People's Hospital, Chongqing, 400070, P. R. China;
2. Department of Orthopedics, the Second Affiliated Hospital of Chongqing Medical University;

Corresponding author：

TONGWei, Email: 25207219@qq.com

Keywords：

Sciatic nerve injury; Nerve growth factor; Myelin associated glycoprotein; Adenovirus; Nerve repair; Gene therapy; Rat

DOI：

10.7507/1002-1892.20160206

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Objective To construct recombinant adenovirus expressing nerve growth factor (NGF) and myelin associated glycoprotein (MAG) (Ad-NGF-MAG) and to investigate its effect on repair and regeneration of sciatic nerve injury in rats. Methods NGF and MAG gene sequences were cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK293 cells, the recombinant Ad-NGF-MAG underwent sequence and identification. Thirty-two male Sprague Dawley rats were randomly divided into 4 groups (n=8): control group (normal control), adenovirus vector group (Ad group), Ad-NGF group, and Ad-NGF-MAG group. The sciatic nerve injury model was established by transection of the right sciatic nerve; then, the empty adenovirus vector, Ad-NGF, and Ad-NGF-MAG were injected into the gastrocnemius muscle of the affected limb at a dose of 1×10⁸ PFU every other day for 3 times in Ad group, AdNGF group, and Ad-NGF-MAG group, respectively. The right sciatic nerve was exposed only, and then the incision was closed in the control group. The sciatic nerve function index (SFI) was measured, and neuro-electrophysiology was observed; mRNA and protein expressions of NGF and MAG were detected by RT-PCR and Western blot; and histological examination was performed at 31 days after operation. Results Recombinant adenovirus vectors of Ad-NGF and Ad-NGF-MAG were constructed successfully. All rats survived and incision healed by first intension. The SFI, nerve conduction velocity, evoked potential amplitude, and latent period of Ad-NGF-MAG group were significantly better than those of Ad group and Ad-NGF group (P < 0.05). MAG mRNA and protein expressions of Ad-NGF-MAG group were the highest in all the groups (P < 0.05). The expressions of NGF mRNA and protein increased in Ad-NGF group and AdNGF-MAG group when compared with control group and Ad group (P < 0.05). Histological examination showed that the nerve had good continuity in control group; nerve fibers disarranged in Ad group; neurons connections formed in some nerve fibers of Ad-NGF group, but nerve fibers arrange disorderly; and the growth of the nerve were ordered and wellstructured in Ad-NGF-MAG group. Conclusion Ad-NGF-MAG can effectively promote the growth of the nerve and inhibit the form of abnormal branches, facilitating the repair of sciatic nerve injury in rats.

Citation： CHENYu, DENGZhongliang, WENGZheng, CHENShimou, TONGWei. EFFECT OF RECOMBINANT CO-EXPRESSION ADENOVIRUS OF NERVE GROWTH FACTOR AND MYELIN ASSOCIATED GLYCOPROTEIN GENES ON RAT SCIATIC NERVE INJURY. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(8): 1026-1033. doi: 10.7507/1002-1892.20160206 Copy

1.	Gupta R, Steward O. Chronic nerve compression induces concurrent apoptosis and proliferation of Schwann cells. J Comp Neurol, 2003, 461(2):174-186.
2.	Tomita K, Kubo T, Matsuda K, et al. Myelin-associated glycoprotein reduces axonal branching and enhances functional recovery after sciatic nerve transection in rats. Glia, 2007, 55(14):1498-1507.
3.	Micera A, Puxeddu I, Aloe L, et al. New insights on the involvement of nerve growth factor in a1lergic inflammation and fibrosis. Cytokine Growth Factor Rev, 2003, 14(5):369-374.
4.	陈渝, 王大勇, 王祖贵, 等.携带NGF基因腺病毒治疗大鼠坐骨神经损伤的实验研究.中国修复重建外科杂志, 2009, 23(8):947-953.
5.	Nakajima H, Uchida K, Kobayashi S, et al. Targeted retrograde gene delivery into the injured cervical spinal cord using recombinant adenovirus vector. Neurosci Lett, 2005, 385(1):30-35.
6.	Zhu JY, Huang YT, Zhu QS, et al. Expression of adenovirusmediated neurotrophin-3 gene in Schwann cells of sciatic nerve in rats. Chin J Traumatol, 2003, 6(2):75-80.
7.	Cao Z, Qiu J, Domemconi M, et al. The inhibition site on myelinassociated glycoprotein is within Ig-domain 5 and is distinct from the sialic acid binding site. J Neurosci, 2007, 27(34):9146-9154.
8.	DeBellard ME, Tang S, Mukhopadhyay G, et al. Myelin-associated glycoplotein inhibits axonal regeneration from a variety of neurons via interaction with a sialoglycoprotein. MoI Cell Neurosci, 1996, 7(2):89-101.
9.	Quarles RH. Myelin-associated glycoprotein (MAG):past, present and beyond. J Neurochem, 2007, 100(6):1431-1448.
10.	Cao Z, Gao Y, Deng K, et al. Receptors for myelin inhibitors: Structures and therapeutic opportunities. Mol Cell Neurosci, 2010, 43(1):1-14.
11.	McLean J, Batt J, Doering LC, et al. Enhanced rate of nerve regeneration and directional errors after sciatic nerve injury in receptor protein tyrosine phosphatase sigma knock-out mice. J Neurosci, 2002, 22(13):5481-5491.
12.	Mukhopadhyay G, Doherty P, Welsh FS, et al. A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron, 1994, 13(3):757-767.
13.	Quarles RH. A hypothesis about the relationship of myelinassociated glycoprotein's function in myelinated axons to its capacity to inhibit neurite outgrowth. Neurochem Res, 2009, 34(1): 79-86.
14.	Aloe L. Rita Levi-Montalcini and the discovery of nerve growth factor:past and present studies. Arch Ital Biol, 2003, 141(2-3):65-83.
15.	Miller C, Jeftinija S, Mallapragada S. Synergistic effects of physical and chemical guidance cues on neurite alignment and outgrowth on biodegradable polymer substrates. Tissue Eng, 2002, 8(3):367-78.
16.	Lesný P, De Croos J, Prádný M, et al. Polymer hydrogels usable for nervous tissue repair. J Chem Neuroanat, 2002, 23(4):243-247.
17.	Miller C, Jeftinija S, Mallapragada S. Micropatterned Schwann cellseeded biodegradable polymer substrates significantly enhance neurite alignment and outgrowth. Tissue Eng, 2001, 7(6):705-715.
18.	Filbin MT. Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat Rev Neurosci, 2003, 4(9):703-713.

1. Gupta R, Steward O. Chronic nerve compression induces concurrent apoptosis and proliferation of Schwann cells. J Comp Neurol, 2003, 461(2):174-186.
2. Tomita K, Kubo T, Matsuda K, et al. Myelin-associated glycoprotein reduces axonal branching and enhances functional recovery after sciatic nerve transection in rats. Glia, 2007, 55(14):1498-1507.
3. Micera A, Puxeddu I, Aloe L, et al. New insights on the involvement of nerve growth factor in a1lergic inflammation and fibrosis. Cytokine Growth Factor Rev, 2003, 14(5):369-374.
4. 陈渝, 王大勇, 王祖贵, 等.携带NGF基因腺病毒治疗大鼠坐骨神经损伤的实验研究.中国修复重建外科杂志, 2009, 23(8):947-953.
5. Nakajima H, Uchida K, Kobayashi S, et al. Targeted retrograde gene delivery into the injured cervical spinal cord using recombinant adenovirus vector. Neurosci Lett, 2005, 385(1):30-35.
6. Zhu JY, Huang YT, Zhu QS, et al. Expression of adenovirusmediated neurotrophin-3 gene in Schwann cells of sciatic nerve in rats. Chin J Traumatol, 2003, 6(2):75-80.
7. Cao Z, Qiu J, Domemconi M, et al. The inhibition site on myelinassociated glycoprotein is within Ig-domain 5 and is distinct from the sialic acid binding site. J Neurosci, 2007, 27(34):9146-9154.
8. DeBellard ME, Tang S, Mukhopadhyay G, et al. Myelin-associated glycoplotein inhibits axonal regeneration from a variety of neurons via interaction with a sialoglycoprotein. MoI Cell Neurosci, 1996, 7(2):89-101.
9. Quarles RH. Myelin-associated glycoprotein (MAG):past, present and beyond. J Neurochem, 2007, 100(6):1431-1448.
10. Cao Z, Gao Y, Deng K, et al. Receptors for myelin inhibitors: Structures and therapeutic opportunities. Mol Cell Neurosci, 2010, 43(1):1-14.
11. McLean J, Batt J, Doering LC, et al. Enhanced rate of nerve regeneration and directional errors after sciatic nerve injury in receptor protein tyrosine phosphatase sigma knock-out mice. J Neurosci, 2002, 22(13):5481-5491.
12. Mukhopadhyay G, Doherty P, Welsh FS, et al. A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron, 1994, 13(3):757-767.
13. Quarles RH. A hypothesis about the relationship of myelinassociated glycoprotein's function in myelinated axons to its capacity to inhibit neurite outgrowth. Neurochem Res, 2009, 34(1): 79-86.
14. Aloe L. Rita Levi-Montalcini and the discovery of nerve growth factor:past and present studies. Arch Ital Biol, 2003, 141(2-3):65-83.
15. Miller C, Jeftinija S, Mallapragada S. Synergistic effects of physical and chemical guidance cues on neurite alignment and outgrowth on biodegradable polymer substrates. Tissue Eng, 2002, 8(3):367-78.
16. Lesný P, De Croos J, Prádný M, et al. Polymer hydrogels usable for nervous tissue repair. J Chem Neuroanat, 2002, 23(4):243-247.
17. Miller C, Jeftinija S, Mallapragada S. Micropatterned Schwann cellseeded biodegradable polymer substrates significantly enhance neurite alignment and outgrowth. Tissue Eng, 2001, 7(6):705-715.
18. Filbin MT. Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat Rev Neurosci, 2003, 4(9):703-713.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF RECOMBINANT CO-EXPRESSION ADENOVIRUS OF NERVE GROWTH FACTOR AND MYELIN ASSOCIATED GLYCOPROTEIN GENES ON RAT SCIATIC NERVE INJURY

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