骨髓源性細胞促進坐骨神經體外預變性實驗研究_《中國修復重建外科雜志》

作者：

吳敏 , 王曉盼 , 肖玉周 , 周建生

蚌埠醫學院第一附屬醫院骨科組織移植安徽省重點實驗室（安徽蚌埠，233003）;

關鍵詞：

坐骨神經雪旺細胞骨髓源性細胞體外預變性小鼠

DOI：

10.7507/1002-1892.20130123

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目的探討周圍神經體外預變性的新方法，以便在短期內獲得大量高效雪旺細胞，以期為組織工程神經構建提供大量種子細胞。方法采用轉綠色熒光蛋白基因C57BL/6小鼠的骨髓源性細胞（bone marrow derived cells，BMDCs）與C57BL/6小鼠坐骨神經體外共培養，建立體外預變性模型的實驗組（A組），無BMDCs參與的單純坐骨神經體外培養為對照組（B組）。培養7 d后行大體觀察及免疫熒光染色觀察BMDCs能否在體外進入坐骨神經內參與預變性；將變性后的神經酶消化行雪旺細胞培養，通過細胞免疫熒光染色及流式細胞儀檢測各組細胞量及鑒定原代培養后的雪旺細胞純度，評價各組雪旺細胞增殖情況。結果培養7 d后大體觀察示兩組坐骨神經斷端開始形成神經瘤樣結構，A組較B組明顯；免疫熒光染色示A組大量BMDCs浸潤至神經內部，其中部分細胞表達F4/80，為單核巨噬系統細胞。經細胞培養，A、B組獲得的雪旺細胞產量分別為（5.59 ± 0.19）× 104個/mg和（3.20 ± 0.21） × 104個/mg，差異有統計學意義（t=2.14，P=0.03）。細胞接種后48 h經p75NTR熒光染色鑒定示，兩組可見雙極或三極樣雪旺細胞，細胞核為藍色且較小，胞體為紅色；成纖維細胞呈扁平多角形，細胞核及核仁清晰，大而不透光，多位于雪旺細胞下且p75NTR染色為陰性。A組混雜的成纖維細胞較少，B組較多。A、B組雪旺細胞純度分別為88.4% ± 5.8%和76.1% ± 3.7%，差異有統計學意義（t=2.38，P=0.04）。經流式細胞儀定量分析A組雪旺細胞純度為89.6%，B組為74.9%。結論BMDCs與周圍神經體外共培養是一種有效獲得大量雪旺細胞的方法，為組織工程神經構建中種子細胞的獲取提供了新方法。

引用本文： 吳敏,王曉盼,肖玉周,周建生. 骨髓源性細胞促進坐骨神經體外預變性實驗研究. 中國修復重建外科雜志, 2013, 27(5): 554-558. doi: 10.7507/1002-1892.20130123 復制

1.	Barrette B, Hebert MA, Filali M, et al. Requirement of myeloid cells for axon regeneration. J Neurosci, 2008, 28(38): 9363-9376.
2.	Lee H, Jo EK, Choi SY, et al. Necrotic neuronal cells induce inflammatory Schwann cell activation via TLR2 and TLR3: implication in Wallerian degeneration. Biochem Biophys Res Commun, 2006, 350(3): 742-747.
3.	Boivin A, Pineau I, Barrette B, et al. Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. J Neurosci, 2007, 27(46): 12565-12576.
4.	Stoll G, Jander S, Myers RR. Degeneration and regeneration of the peripheral nervous system: from Augustus Waller’s observations to neuroinflammation. J Peripher Nerv Syst, 2002, 7(1): 13-27.
5.	Yang DP, Zhang DP, Mak KS, et al. Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis. Mol Cell Neurosci, 2008, 38(1): 80-88.
6.	Dahlin LB. Prevention of macrophage invasion impairs regeneration in nerve grafts. Brain Res, 1995, 679(2): 274-280.
7.	Barrette B, Hébert MA, Filali M, et al. Requirement of myeloid cells for axon regeneration. J Neurosci, 2008, 28(38): 9363-9376.
8.	Gensel JC, Nakamura S, Guan Z, et al. Macrophages promote axon regeneration with concurrent neurotoxicity. J Neurosci, 2009, 29(12): 3956-3968.
9.	Ide C. Peripheral nerve regeneration. Neurosci Res, 1996, 25(2): 101-121.
10.	Frostick SP, Yin Q, Kemp GJ. Schwann cells, neurotrophic factors, and peripheral nerve regeneration. Microsurgery, 1998, 18(7): 397-405.
11.	Keilhoff G, Fansa H, Schneider W, et al. In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits. J Neurosci Methods, 1999, 89(1): 17-24.
12.	Kraus A, Täger J, Kohler K, et al. Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures. J Neurotrauma, 2010, 27(1): 197-203.
13.	Lindholm D, Heumann R, Meyer M, et al. Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature, 1987, 330(6149): 658-659.
14.	Lindholm D, Heumann R, Hengerer B, et al. Interleukin 1 increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts. J Biol Chem, 1988, 263(31): 16348-16351.
15.	Santala P, Heino J. Regulation of integrin-type cell adhesion receptors by cytokines. J Biol Chem, 1991, 266(34): 23505-23509.
16.	Taskinen HS, Heino J, Röyttä M. The dynamics of beta 1 integrin expression during peripheral nerve regeneration. Acta Neuropathol, 1995, 89(2): 144-151.
17.	Ridley AJ, Davis JB, Stroobant P, et al. Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells. J Cell Biol, 1989, 109(6 Pt 2): 3419-3424.
18.	Stoll G, Griffin JW, Li CY, et al. Wallerian degeneration in the peripheral nervous system: participation of both Schwann cells and macrophages in myelin degradation. J Neurocytol, 1989, 18(5): 671-683.
19.	Miranda CO, Teixeira CA, Liz MA, et al. Systemic delivery of bone marrow-derived mesenchymal stromal cells diminishes neuropathology in a mouse model of Krabbe’s disease. Stem Cells, 2011, 29(11): 1738-1751.
20.	Zuo J, Hernandez YJ, Muir D. Chondroitin sulfate proteoglycan with neurite-inhibiting activity is up-regulated following peripheral nerve injury. J Neurobiol, 1998, 34(1): 41-54.
21.	Zuo J, Ferguson TA, Hernandez YJ, et al. Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan. J Neurosci, 1998, 18(14): 5203-5211.
22.	Ferguson TA, Muir D. MMP-2 and MMP-9 increase the neurite-promoting potential of schwann cell basal laminae and are upregulated in degenerated nerve. Mol Cell Neurosci, 2000, 16(2): 157-167.
23.	Yamada T, Miyazaki K, Koshikawa N, et al. Selective localization of gelatinase A, an enzyme degrading beta-amyloid protein, in white matter microglia and in Schwann cells. Acta Neuropathol, 1995, 89(3): 199-203.
24.	Krekoski CA, Neubauer D, Graham JB, et al. Metalloproteinase-dependent predegeneration in vitro enhances axonal regeneration within acellular peripheral nerve grafts. J Neurosci, 2002, 22(23): 10408-10415.
25.	Siebert H, Dippel N, Mäder M, et al. Matrix metalloproteinase expression and inhibition after sciatic nerve axotomy. J Neuropathol Exp Neurol, 2001, 60(1): 85-93.
26.	Shubayev VI, Myers RR. Upregulation and interaction of TNFalpha and gelatinases A and B in painful peripheral nerve injury. Brain Res, 2000, 855(1): 83-89.

1. Barrette B, Hebert MA, Filali M, et al. Requirement of myeloid cells for axon regeneration. J Neurosci, 2008, 28(38): 9363-9376.
2. Lee H, Jo EK, Choi SY, et al. Necrotic neuronal cells induce inflammatory Schwann cell activation via TLR2 and TLR3: implication in Wallerian degeneration. Biochem Biophys Res Commun, 2006, 350(3): 742-747.
3. Boivin A, Pineau I, Barrette B, et al. Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. J Neurosci, 2007, 27(46): 12565-12576.
4. Stoll G, Jander S, Myers RR. Degeneration and regeneration of the peripheral nervous system: from Augustus Waller’s observations to neuroinflammation. J Peripher Nerv Syst, 2002, 7(1): 13-27.
5. Yang DP, Zhang DP, Mak KS, et al. Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis. Mol Cell Neurosci, 2008, 38(1): 80-88.
6. Dahlin LB. Prevention of macrophage invasion impairs regeneration in nerve grafts. Brain Res, 1995, 679(2): 274-280.
7. Barrette B, Hébert MA, Filali M, et al. Requirement of myeloid cells for axon regeneration. J Neurosci, 2008, 28(38): 9363-9376.
8. Gensel JC, Nakamura S, Guan Z, et al. Macrophages promote axon regeneration with concurrent neurotoxicity. J Neurosci, 2009, 29(12): 3956-3968.
9. Ide C. Peripheral nerve regeneration. Neurosci Res, 1996, 25(2): 101-121.
10. Frostick SP, Yin Q, Kemp GJ. Schwann cells, neurotrophic factors, and peripheral nerve regeneration. Microsurgery, 1998, 18(7): 397-405.
11. Keilhoff G, Fansa H, Schneider W, et al. In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits. J Neurosci Methods, 1999, 89(1): 17-24.
12. Kraus A, Täger J, Kohler K, et al. Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures. J Neurotrauma, 2010, 27(1): 197-203.
13. Lindholm D, Heumann R, Meyer M, et al. Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature, 1987, 330(6149): 658-659.
14. Lindholm D, Heumann R, Hengerer B, et al. Interleukin 1 increases stability and transcription of mRNA encoding nerve growth factor in cultured rat fibroblasts. J Biol Chem, 1988, 263(31): 16348-16351.
15. Santala P, Heino J. Regulation of integrin-type cell adhesion receptors by cytokines. J Biol Chem, 1991, 266(34): 23505-23509.
16. Taskinen HS, Heino J, Röyttä M. The dynamics of beta 1 integrin expression during peripheral nerve regeneration. Acta Neuropathol, 1995, 89(2): 144-151.
17. Ridley AJ, Davis JB, Stroobant P, et al. Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells. J Cell Biol, 1989, 109(6 Pt 2): 3419-3424.
18. Stoll G, Griffin JW, Li CY, et al. Wallerian degeneration in the peripheral nervous system: participation of both Schwann cells and macrophages in myelin degradation. J Neurocytol, 1989, 18(5): 671-683.
19. Miranda CO, Teixeira CA, Liz MA, et al. Systemic delivery of bone marrow-derived mesenchymal stromal cells diminishes neuropathology in a mouse model of Krabbe’s disease. Stem Cells, 2011, 29(11): 1738-1751.
20. Zuo J, Hernandez YJ, Muir D. Chondroitin sulfate proteoglycan with neurite-inhibiting activity is up-regulated following peripheral nerve injury. J Neurobiol, 1998, 34(1): 41-54.
21. Zuo J, Ferguson TA, Hernandez YJ, et al. Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan. J Neurosci, 1998, 18(14): 5203-5211.
22. Ferguson TA, Muir D. MMP-2 and MMP-9 increase the neurite-promoting potential of schwann cell basal laminae and are upregulated in degenerated nerve. Mol Cell Neurosci, 2000, 16(2): 157-167.
23. Yamada T, Miyazaki K, Koshikawa N, et al. Selective localization of gelatinase A, an enzyme degrading beta-amyloid protein, in white matter microglia and in Schwann cells. Acta Neuropathol, 1995, 89(3): 199-203.
24. Krekoski CA, Neubauer D, Graham JB, et al. Metalloproteinase-dependent predegeneration in vitro enhances axonal regeneration within acellular peripheral nerve grafts. J Neurosci, 2002, 22(23): 10408-10415.
25. Siebert H, Dippel N, Mäder M, et al. Matrix metalloproteinase expression and inhibition after sciatic nerve axotomy. J Neuropathol Exp Neurol, 2001, 60(1): 85-93.
26. Shubayev VI, Myers RR. Upregulation and interaction of TNFalpha and gelatinases A and B in painful peripheral nerve injury. Brain Res, 2000, 855(1): 83-89.

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骨髓源性細胞促進坐骨神經體外預變性實驗研究

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