羧甲基殼聚糖對體外培養雪旺細胞增殖周期及其分泌神經營養因子的影響_《中國修復重建外科雜志》

作者：

賀斌 , 陶海鷹 , 衛愛林 , 劉世清

武漢大學人民醫院骨科（武漢，430060）;

關鍵詞：

羧甲基殼聚糖雪旺細胞細胞增殖神經營養因子大鼠

DOI：

10.7507/1002-1892.20130203

視頻：

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目的探討羧甲基殼聚糖（carboxymethylated chitosan，CMCS）對雪旺細胞（Schwann cells，SCs）增殖周期及分泌神經營養因子的影響及其作用機制。方法取3～5日齡SPF級SD大鼠20只，雌雄不限，體重25～30 g，取雙側坐骨神經體外培養SCs，經S-100免疫熒光標記鑒定。取處于對數生長期的第2代SCs，細胞計數檢測試劑盒8檢測不同濃度CMCS及作用不同時間對SCs增殖的影響。將SCs分為4組，加入CMCS調整終濃度為50（B組）、100（C組）、200 μg/mL（D組），對照組（A組）加入等量PBS，流式細胞技術檢測處于S期細胞的百分比（S%）和增殖指數（proliferation index，PI）分析細胞周期；實時定量PCR與Western blot檢測SCs合成NGF和睫狀神經營養因子（ciliary neurotrophic factor，CNTF）的含量。結果S-100免疫熒光標記鑒定示細胞陽性率達90%以上。CMCS在10～1 000 μg/ mL濃度范圍內對SCs增殖均有促進作用，200、500 μg/mL組促增殖效應最明顯（P lt; 0.01），200 μg/mL與500 μg/mL組間比較差異無統計學意義（P gt; 0.05）。采用200 μg/mL CMCS培養SCs不同時間，發現作用24 h時其促增殖效果最明顯，與對照組（200 μg/mL CMCS作用0 h）比較差異有統計學意義（P lt; 0.01）。B、C、D組S%及PI均顯著高于A組，差異有統計學意義（P lt; 0.05）；C、D組顯著高于B組（P lt; 0.05），C、D組間差異無統計學意義（P gt; 0.05）。實時定量PCR和Western blot檢測均顯示，B、C、D組NGF與CNTF mRNA和蛋白表達均顯著高于A組（P lt; 0.05）；其中D組作用最明顯。結論CMCS可以促進SCs增殖及細胞周期進程以及分泌神經營養因子。

引用本文： 賀斌,陶海鷹,衛愛林,劉世清. 羧甲基殼聚糖對體外培養雪旺細胞增殖周期及其分泌神經營養因子的影響. 中國修復重建外科雜志, 2013, 27(8): 923-927. doi: 10.7507/1002-1892.20130203 復制

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.	賀斌, 劉世清, 陳慶, 等. 羧甲基殼聚糖對體外培養髓核細胞增殖及硝普鈉誘導凋亡的影響. 中國矯形外科雜志, 2011, 19(13): 1126-1129.
9.	陶海鷹, 賀斌, 衛愛林, 等. 羧甲基殼聚糖對硝普鈉誘導大鼠髓核細胞誘導型一氧化氮合酶和基質金屬蛋白酶表達的影響. 中華實驗外科雜志, 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, 陸國才, 劉俊平, 等. 神經生長因子對擠壓傷坐骨神經再生的促進作用. 中國臨床康復, 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. 賀斌, 劉世清, 陳慶, 等. 羧甲基殼聚糖對體外培養髓核細胞增殖及硝普鈉誘導凋亡的影響. 中國矯形外科雜志, 2011, 19(13): 1126-1129.
9. 陶海鷹, 賀斌, 衛愛林, 等. 羧甲基殼聚糖對硝普鈉誘導大鼠髓核細胞誘導型一氧化氮合酶和基質金屬蛋白酶表達的影響. 中華實驗外科雜志, 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, 陸國才, 劉俊平, 等. 神經生長因子對擠壓傷坐骨神經再生的促進作用. 中國臨床康復, 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.

《中國修復重建外科雜志》

羧甲基殼聚糖對體外培養雪旺細胞增殖周期及其分泌神經營養因子的影響

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羧甲基殼聚糖對體外培養雪旺細胞增殖周期及其分泌神經營養因子的影響

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