EFFECT OF CARBOXYMETHYLATED CHITOSAN ON PROLIFERATION AND SYNTHESIS OF NEUROTROPHIC FACTORS IN Schwann CELLS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HE Bin , TAO Haiying , WEI Ailin , LIU Shiqing.

Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan Hubei, 430060, P.R.China. Corresponding author: HE Bin, E-mail: hebin@whu.edu.cn;

Corresponding author：

Keywords：

Carboxymethylated chitosan; Schwann cells; Cell proliferation; Neurotrophic factor; Rat

DOI：

10.7507/1002-1892.20130203

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Objective To investigate the effect of carboxymethylated chitosan (CMCS) on the proliferation, cell cycle, and secretion of neurotrophic factors in cultured Schwann cells (SCs). Methods SCs were obtained from sciatic nerves of 20 Sprague Dawley rats (3-5 days old; male or female; weighing, 25-30 g) and cultured in vitro, SCs were identified and purified by immunofluorescence against S-100. The cell counting kit 8 (CCK-8) assay was used to determine the proliferation of SCs. The SCs were divided into 4 groups: 50 μg/mL CMCS (group B), 100 μg/mL CMCS (group C), 200 μg/mL CMCS (group D), and the same amount of PBS (group A) were added. The flow cytometry was used to analyze the cell cycle of SCs; the real-time quantitative PCR and Western blot analysis were used to detect the levels of never growth factor (NGF) and ciliary neurotrophic factor (CNTF) in cultured SCs induced by CMCS. Results The purity of cultured SCs was more than 90% by immunofluorescence against S-100; the CCK-8 results indicated that CMCS in concentrations of 10-1 000 μg/mL could promote the proliferation of SCs, especially in concentrations of 200 and 500 μg/mL (P lt; 0.01), but no significant difference was found between 200 and 500 μg/mL (P gt; 0.05). CMCS at a concentration of 200 μg/mL for 24 hours induced the highest proliferation, showing significant difference when compared with that at 0 hour (P lt; 0.01). The percentage of cells in phase S and the proliferation index were significantly higher in groups B, C, and D than in group A (P lt; 0.05), in groups C and D than in group B (P lt; 0.05); and there was no significant difference between group C and group D (P gt; 0.05). Real-time quantitative PCR and Western blot results showed that the levels of NGF and CNTF in groups B, C, and D were significantly higher than those in group A (P lt; 0.05), especially in group D. Conclusion CMCS can stimulate the proliferation, and induce the synthesis of neurotrophic factors in cultured SCs.

Citation： HE Bin,TAO Haiying,WEI Ailin,LIU Shiqing.. EFFECT OF CARBOXYMETHYLATED CHITOSAN ON PROLIFERATION AND SYNTHESIS OF NEUROTROPHIC FACTORS IN Schwann CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(8): 923-927. doi: 10.7507/1002-1892.20130203 Copy

1.	Guest JD, Rao A, Olson L, et al. The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord. Exp Neurol, 1997, 148(2): 502-522.
2.	Guseva D, Angelov DN, Irintchev A, et al. Ablation of adhesion molecule L1 in mice favours Schwann cell proliferation and functional recovery after peripheral nerve injury. Brain, 2009, 132(Pt 8): 2180-2195.
3.	Jain S, Webster TJ, Sharma A, et al. Intracellular reactive oxidative stress, cell proliferation and apoptosis of Schwann cells on carbon nanofibrous substrates. Biomaterials, 2013, 34(21): 4891-4901.
4.	Chen Z, Chang R, Guan W, et al. Transplantation of porcine hepatocytes cultured with polylactic Acid-o-carboxymethylated chitosan nanoparticles promotes liver regeneration in acute liver failure rats. J Drug Deliv, 2011, 2011: 797503.
5.	Wang F, Chen Y, Zhang D, et al. Folate-mediated targeted and intracellular delivery of paclitaxel using a novel deoxycholic acid-O-carboxymethylated chitosan-folic acid micelles. Int J Nanomedicine, 2012, 7: 325-337.
6.	Chen Q, Liu SQ, Du YM, et al. Carboxymethyl-chitosan protects rabbit chondrocytes from interleukin-1beta-induced apoptosis. Eur J Pharmacol, 2006, 541(1-2): 1-8.
7.	Liu SQ, Qiu B, Chen LY, et al. The effects of carboxymethylated chitosan on metalloproteinase-1, 3 and tissue inhibitor of metalloproteinase-1 gene expression in cartilage of experimental osteoarthritis. Rheumatol Int, 2005, 26(1): 52-57.
8.	贺斌, 刘世清, 陈庆, 等. 羧甲基壳聚糖对体外培养髓核细胞增殖及硝普钠诱导凋亡的影响. 中国矫形外科杂志, 2011, 19(13): 1126-1129.
9.	陶海鹰, 贺斌, 卫爱林, 等. 羧甲基壳聚糖对硝普钠诱导大鼠髓核细胞诱导型一氧化氮合酶和基质金属蛋白酶表达的影响. 中华实验外科杂志, 2012, 29(12): 2545-2548.
10.	He B, Liu SQ, Chen Q, et al. Carboxymethylated chitosan stimulates proliferation of Schwann cells in vitro via the activation of the ERK and Akt signaling pathways. Eur J Pharmacol, 2011, 667(1-3): 195-201.
11.	Tao HY, He B, Liu SQ, et al. Effect of carboxymethylated chitosan on the biosysthesis of NGF and activation of the Wnt/β-catenin signaling pathway in the proliferation of Schwann cells. Eur J Pharmacol, 2013, 702(1-3): 85-92.
12.	Brockes JP, Fields KL, Raff MC. Studies on cultured rat Schwann cells. I. Establishment of purified population from cultures of peripheral nerve. Brain Res, 1979, 165(1): 105-118.
13.	Jin YQ, Liu W, Hong TH, et al. Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment. J Neurosci Methods, 2008, 170(1): 140-148.
14.	Keyvan FN, Raisman G, Li Y, et al. Delayed repair of corticospinal tract lesions as an assay for the effectiveness of transplantation of Schwann cells. Glia, 2005, 51(4): 306-311.
15.	Murray-Dunning C, McArthur SL, Sun T, et al. Three-dimensional alignment of Schwann cells using hydrolysable microfiber scaffolds: strategies for peripheral nerve repair. Methods Mol Biol, 2011, 695: 155-166.
16.	Udina E, Ceballos D, Gold BG, et al. FK506 enhances reinnervations by regeneration and collateral sprouting of peripheral nerve fibers. Exp Neurol, 2003, 183(1): 220-231.
17.	Chen YS, Murakami S, Gyo K, et al. Effects of basic fibroblast growth factor (bFGF)-neuralizing antibody and platelet factor 4 on facial nerve regeneration. Exp Neurol, 1999, 155(2): 274-283.
18.	De Nicolo S, Tarani L, Ceccanti M, et al. Effects of olive polyphenols administration on nerve growth factor and brain-derived neurotrophic factor in the mouse brain. Nutrition, 2013, 29(4): 681-687.
19.	Kaechi K, Ikegami R, Nakamura N, et al. 4-Methylcatechol, an inducer of nerve growth factor synthesis, enhances peripheral nerve regeneration across nerve gaps. J Pharmacol Exp Ther, 1995, 272(3): 1300-1304.
20.	袁伯俊, 陆国才, 刘俊平, 等. 神经生长因子对挤压伤坐骨神经再生的促进作用. 中国临床康复, 2005, 9(5): 178-180.
21.	Zhang J, Lineaweaver WC, Oswald T, et al. Ciliary nrurotrophic factor for acceleration of peripheral nerve regeneration: an experimental study. Reconstr Microsurg, 2004, 20(4): 323-327.

1. Guest JD, Rao A, Olson L, et al. The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord. Exp Neurol, 1997, 148(2): 502-522.
2. Guseva D, Angelov DN, Irintchev A, et al. Ablation of adhesion molecule L1 in mice favours Schwann cell proliferation and functional recovery after peripheral nerve injury. Brain, 2009, 132(Pt 8): 2180-2195.
3. Jain S, Webster TJ, Sharma A, et al. Intracellular reactive oxidative stress, cell proliferation and apoptosis of Schwann cells on carbon nanofibrous substrates. Biomaterials, 2013, 34(21): 4891-4901.
4. Chen Z, Chang R, Guan W, et al. Transplantation of porcine hepatocytes cultured with polylactic Acid-o-carboxymethylated chitosan nanoparticles promotes liver regeneration in acute liver failure rats. J Drug Deliv, 2011, 2011: 797503.
5. Wang F, Chen Y, Zhang D, et al. Folate-mediated targeted and intracellular delivery of paclitaxel using a novel deoxycholic acid-O-carboxymethylated chitosan-folic acid micelles. Int J Nanomedicine, 2012, 7: 325-337.
6. Chen Q, Liu SQ, Du YM, et al. Carboxymethyl-chitosan protects rabbit chondrocytes from interleukin-1beta-induced apoptosis. Eur J Pharmacol, 2006, 541(1-2): 1-8.
7. Liu SQ, Qiu B, Chen LY, et al. The effects of carboxymethylated chitosan on metalloproteinase-1, 3 and tissue inhibitor of metalloproteinase-1 gene expression in cartilage of experimental osteoarthritis. Rheumatol Int, 2005, 26(1): 52-57.
8. 贺斌, 刘世清, 陈庆, 等. 羧甲基壳聚糖对体外培养髓核细胞增殖及硝普钠诱导凋亡的影响. 中国矫形外科杂志, 2011, 19(13): 1126-1129.
9. 陶海鹰, 贺斌, 卫爱林, 等. 羧甲基壳聚糖对硝普钠诱导大鼠髓核细胞诱导型一氧化氮合酶和基质金属蛋白酶表达的影响. 中华实验外科杂志, 2012, 29(12): 2545-2548.
10. He B, Liu SQ, Chen Q, et al. Carboxymethylated chitosan stimulates proliferation of Schwann cells in vitro via the activation of the ERK and Akt signaling pathways. Eur J Pharmacol, 2011, 667(1-3): 195-201.
11. Tao HY, He B, Liu SQ, et al. Effect of carboxymethylated chitosan on the biosysthesis of NGF and activation of the Wnt/β-catenin signaling pathway in the proliferation of Schwann cells. Eur J Pharmacol, 2013, 702(1-3): 85-92.
12. Brockes JP, Fields KL, Raff MC. Studies on cultured rat Schwann cells. I. Establishment of purified population from cultures of peripheral nerve. Brain Res, 1979, 165(1): 105-118.
13. Jin YQ, Liu W, Hong TH, et al. Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment. J Neurosci Methods, 2008, 170(1): 140-148.
14. Keyvan FN, Raisman G, Li Y, et al. Delayed repair of corticospinal tract lesions as an assay for the effectiveness of transplantation of Schwann cells. Glia, 2005, 51(4): 306-311.
15. Murray-Dunning C, McArthur SL, Sun T, et al. Three-dimensional alignment of Schwann cells using hydrolysable microfiber scaffolds: strategies for peripheral nerve repair. Methods Mol Biol, 2011, 695: 155-166.
16. Udina E, Ceballos D, Gold BG, et al. FK506 enhances reinnervations by regeneration and collateral sprouting of peripheral nerve fibers. Exp Neurol, 2003, 183(1): 220-231.
17. Chen YS, Murakami S, Gyo K, et al. Effects of basic fibroblast growth factor (bFGF)-neuralizing antibody and platelet factor 4 on facial nerve regeneration. Exp Neurol, 1999, 155(2): 274-283.
18. De Nicolo S, Tarani L, Ceccanti M, et al. Effects of olive polyphenols administration on nerve growth factor and brain-derived neurotrophic factor in the mouse brain. Nutrition, 2013, 29(4): 681-687.
19. Kaechi K, Ikegami R, Nakamura N, et al. 4-Methylcatechol, an inducer of nerve growth factor synthesis, enhances peripheral nerve regeneration across nerve gaps. J Pharmacol Exp Ther, 1995, 272(3): 1300-1304.
20. 袁伯俊, 陆国才, 刘俊平, 等. 神经生长因子对挤压伤坐骨神经再生的促进作用. 中国临床康复, 2005, 9(5): 178-180.
21. Zhang J, Lineaweaver WC, Oswald T, et al. Ciliary nrurotrophic factor for acceleration of peripheral nerve regeneration: an experimental study. Reconstr Microsurg, 2004, 20(4): 323-327.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF CARBOXYMETHYLATED CHITOSAN ON PROLIFERATION AND SYNTHESIS OF NEUROTROPHIC FACTORS IN Schwann CELLS IN VITRO

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