單酶消化復合植塊法培養雪旺細胞的實驗研究_《中國修復重建外科雜志》

作者：

秦義武 ¹ , 趙勁民 ¹ , 蘇偉 ¹ , 李曉峰 ¹ , 羅世興 ² , 劉軍廷 ¹

1廣西醫科大學第一附屬醫院創傷骨科手外科（南寧，530021）;;
2北海市人民醫院骨科;

關鍵詞：

雪旺細胞坐骨神經細胞培養周圍神經修復大鼠

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目的探討一種快速獲取高純度、高活性雪旺細胞的分離、培養方法，并從轉錄及翻譯水平對獲取細胞進行鑒定。方法取4周齡SD大鼠雙側坐骨神經，膠原酶Ⅰ消化15 min后接種于培養瓶中，進行體外培養并按1∶2比例傳代，倒置相差顯微鏡觀察細胞生長狀態及形態變化，MTT法檢測細胞增殖情況并繪制生長曲線，RT-PCR檢測S100及膠原纖維酸性蛋白（glial fibrillary acidic protein，GFAP）基因表達情況，并行免疫組織化學染色觀察S100及GFAP蛋白表達水平，計算細胞純度。結果膠原酶Ⅰ消化后組織塊培養24 h即可見成纖維樣細胞遷出，胞體細長，折光性較弱；48 h后可見大量細胞遷出，細胞多呈雙極或三極，突起細長，胞體飽滿，折光性強，72 h形成細胞集落。細胞傳代后48～72 h即可長滿瓶底，并出現螺旋形細胞集落，多次傳代后細胞仍生長旺盛，細胞質飽滿。MTT結果顯示，第3 代細胞第3天即進入對數生長期，隨時間延長逐漸增殖，第7天進入平臺期，生長曲線呈“S”形。RT-PCR結果示培養細胞表達S100、GFAP基因；免疫組織化學染色檢測顯示大部分細胞呈陽性染色，表達S100及GFAP蛋白，雪旺細胞純度為98.37% ± 0.30%。結論單酶消化復合植塊法可以快速獲得高純度、高活性的大鼠雪旺細胞。

引用本文： 秦義武,趙勁民,蘇偉,李曉峰,羅世興,劉軍廷. 單酶消化復合植塊法培養雪旺細胞的實驗研究. 中國修復重建外科雜志, 2012, 26(9): 1107-1111. doi: 復制

1.	Feneley MR, Fawcett JW, Keynes RJ. The role of Schwann cells in the regeneration of peripheral nerve axons through muscle basal lamina grafts. Exp Neurol, 1991, 114(3): 275-285.
2.	Torigoe K, Tanaka HF, Takahashi A, et al. Basic behavior of migratory Schwann cells in peripheral nerve regeneration. Exp Neurol, 1996, 137(2): 301-308.
3.	Ara J, Bannerman P, Shaheen F, et al. Schwann cell-autonomous role of neuropilin-2. J Neurosci Res, 2005, 79(4): 468-475.
4.	Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997, 14(1-2): 67-116.
5.	Wang JM, Zeng YS, Wu JL, et al. Cograft of neural stem cells and schwann cells overexpressing TrkC and neurotrophin-3 respectively after rat spinal cord transection. Biomaterials, 2011, 32(30): 7454-7468.
6.	Buss A, Pech K, Kakulas BA, et al. Growth-modulating molecules are associated with invading Schwann cells and not astrocytes in human traumatic spinal cord injury. Brain, 2007, 130(Pt 4): 940-953.
7.	Webber CA, Christie KJ, Cheng C, et al. Schwann cells direct peripheral nerve regeneration through the Netrin-1 receptors, DCC and Unc5H2. Glia, 2011, 59(10): 1503-1517.
8.	Tabesh H, Amoabediny G, Nik NS, et al. The role of biodegradable engineered scaffolds seeded with Schwann cells for spinal cord regeneration. Neurochem Int, 2009, 54(2): 73-83.
9.	Jungnickel J, Haastert-Talini K, Claus P, et al. Polysialyltransferase overexpression in Schwann cells mediates different effects during peripheral nerve regeneration. Glycobiology, 2012, 22(1): 107-115.
10.	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.
11.	趙喆, 趙斌, 王玉, 等. 化學去細胞異體神經添加不同組織來源雪旺細胞對周圍神經損傷修復的功能評價. 中國修復重建外科雜志, 2010, 24(11): 1281-1287.
12.	Schmitte R, Tipold A, Stein VM, et al. Genetically modified canine Schwann cells—In vitro and in vivo evaluation of their suitability for peripheral nerve tissue engineering. J Neurosci Methods, 2010, 186(2): 202-208.
13.	McGrath AM, Novikova LN, Novikov LN, et al. BDTM PuraMatrixTM peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration. Brain Res Bull, 2010, 83(5): 207-213.
14.	Kalbermatten DF, Erba P, Mahay D, et al. Schwann cell strip for peripheral nerve repair. J Hand Surg Eur Vol, 2008, 33(5): 587-594.
15.	Bockelmann J, Klinkhammer K, von Holst A, et al. Functionalization of electrospun poly (ε-caprolactone) fibers with the extracellular matrix-derived peptide GRGDS improves guidance of schwann cell migration and axonal growth. Tissue Eng Part A, 2011, 17(3-4): 475-486.
16.	Niapour A, Karamali F, Nemati S, et al. Co-transplantation of human embryonic stem cell-derived neural progenitors and Schwann cells in a rat spinal cord contusion injury model elicits a distinct neurogenesis and functional recovery. Cell Transplant, 2012, 21(5): 827-843.
17.	Askanas V, Engel WK, Dalakas MC, et al. Human schwann cells in tissue culture: histochemical and ultrastructural studies. Arch Neurol, 1980, 37(6): 329-337.
18.	Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J Neurosci Methods, 2003, 122(2): 195-200.
19.	翁慧蘭, 孫淑明, 吳麗娥. 雪旺細胞體外培養和純化的新方法. 中國基層醫藥, 2005, 12(10): 1299-1300.
20.	王世清, 范志海, 沈憶新. 不同部位取材的雪旺細胞培養與純化研究. 中國修復重建外科雜志, 2011, 25(2): 166-170.
21.	劉炎, 張莉, 宮旭, 等. 雙向差速法制備高純度雪旺細胞的實驗研究. 中國矯形外科雜志, 2011, 19(16): 1373-1376.
22.	Graham A. Melanocyte production: dark side of the Schwann cell. Curr Biol, 2009, 19(24): R1116-1117.

1. Feneley MR, Fawcett JW, Keynes RJ. The role of Schwann cells in the regeneration of peripheral nerve axons through muscle basal lamina grafts. Exp Neurol, 1991, 114(3): 275-285.
2. Torigoe K, Tanaka HF, Takahashi A, et al. Basic behavior of migratory Schwann cells in peripheral nerve regeneration. Exp Neurol, 1996, 137(2): 301-308.
3. Ara J, Bannerman P, Shaheen F, et al. Schwann cell-autonomous role of neuropilin-2. J Neurosci Res, 2005, 79(4): 468-475.
4. Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol, 1997, 14(1-2): 67-116.
5. Wang JM, Zeng YS, Wu JL, et al. Cograft of neural stem cells and schwann cells overexpressing TrkC and neurotrophin-3 respectively after rat spinal cord transection. Biomaterials, 2011, 32(30): 7454-7468.
6. Buss A, Pech K, Kakulas BA, et al. Growth-modulating molecules are associated with invading Schwann cells and not astrocytes in human traumatic spinal cord injury. Brain, 2007, 130(Pt 4): 940-953.
7. Webber CA, Christie KJ, Cheng C, et al. Schwann cells direct peripheral nerve regeneration through the Netrin-1 receptors, DCC and Unc5H2. Glia, 2011, 59(10): 1503-1517.
8. Tabesh H, Amoabediny G, Nik NS, et al. The role of biodegradable engineered scaffolds seeded with Schwann cells for spinal cord regeneration. Neurochem Int, 2009, 54(2): 73-83.
9. Jungnickel J, Haastert-Talini K, Claus P, et al. Polysialyltransferase overexpression in Schwann cells mediates different effects during peripheral nerve regeneration. Glycobiology, 2012, 22(1): 107-115.
10. 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.
11. 趙喆, 趙斌, 王玉, 等. 化學去細胞異體神經添加不同組織來源雪旺細胞對周圍神經損傷修復的功能評價. 中國修復重建外科雜志, 2010, 24(11): 1281-1287.
12. Schmitte R, Tipold A, Stein VM, et al. Genetically modified canine Schwann cells—In vitro and in vivo evaluation of their suitability for peripheral nerve tissue engineering. J Neurosci Methods, 2010, 186(2): 202-208.
13. McGrath AM, Novikova LN, Novikov LN, et al. BDTM PuraMatrixTM peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration. Brain Res Bull, 2010, 83(5): 207-213.
14. Kalbermatten DF, Erba P, Mahay D, et al. Schwann cell strip for peripheral nerve repair. J Hand Surg Eur Vol, 2008, 33(5): 587-594.
15. Bockelmann J, Klinkhammer K, von Holst A, et al. Functionalization of electrospun poly (ε-caprolactone) fibers with the extracellular matrix-derived peptide GRGDS improves guidance of schwann cell migration and axonal growth. Tissue Eng Part A, 2011, 17(3-4): 475-486.
16. Niapour A, Karamali F, Nemati S, et al. Co-transplantation of human embryonic stem cell-derived neural progenitors and Schwann cells in a rat spinal cord contusion injury model elicits a distinct neurogenesis and functional recovery. Cell Transplant, 2012, 21(5): 827-843.
17. Askanas V, Engel WK, Dalakas MC, et al. Human schwann cells in tissue culture: histochemical and ultrastructural studies. Arch Neurol, 1980, 37(6): 329-337.
18. Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J Neurosci Methods, 2003, 122(2): 195-200.
19. 翁慧蘭, 孫淑明, 吳麗娥. 雪旺細胞體外培養和純化的新方法. 中國基層醫藥, 2005, 12(10): 1299-1300.
20. 王世清, 范志海, 沈憶新. 不同部位取材的雪旺細胞培養與純化研究. 中國修復重建外科雜志, 2011, 25(2): 166-170.
21. 劉炎, 張莉, 宮旭, 等. 雙向差速法制備高純度雪旺細胞的實驗研究. 中國矯形外科雜志, 2011, 19(16): 1373-1376.
22. Graham A. Melanocyte production: dark side of the Schwann cell. Curr Biol, 2009, 19(24): R1116-1117.

《中國修復重建外科雜志》

單酶消化復合植塊法培養雪旺細胞的實驗研究

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《中國修復重建外科雜志》

單酶消化復合植塊法培養雪旺細胞的實驗研究

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

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Content

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