人胚胎嗅鞘细胞的原代培养及临床意义
摘要
【目的】获得较为理想的取材和纯化嗅鞘细胞的方法措施。
【方法】通过对多种方法的比较获得较为理想纯化嗅鞘细胞的措施，嗅球取材直接挤压法，将细胞接种密度统一调整到10~5／ml后应用3种纯化方法各对10份样本处理并作纯度比较；同样密度和接种数量的原代细胞各取10份种植于3种不同的培养皿，对细胞贴壁速度和最终纯度进行检验。最好的纯化嗅鞘细胞方法为免疫吸附法，其细胞纯度达84.66％。而差速贴壁法(71.59％)和化学试剂法(66.45％)均由于损失细胞太多不可取；同时发现包被培养皿对纯化细胞意义重大，P~(75)包被组不仅培养细胞的纯度最高(88.69％)，而且还有利于OECs的快速贴壁。
【结果】我们培养人胚嗅鞘细胞纯度为84.66％，同时发现包被培养皿对纯化细胞意义重大，p75包被不仅培养细胞的纯度高，而且还有利于OECs的快速贴壁。
【结论】此取材及培养方法收获大量嗅鞘细胞将对其进一步的基础研究以及将来应用于临床奠定基础。
【Objective】To develop the better methods of drawing the materials from OB and purifying OECS.
【Methods】We drawing the materials from OB by crushing directly. We had adjusted inoculated density of OECs to uniform 10~5/ml, We made a contrast of cells purity of each 10 samples disposed by three purification methods respectively; The primary cells which possessed the same density and the same inoculating quantity was fetched 10 portions respectively to implant in three kinds of Petri dish. We checked out cellular anchoring velocity and final purity.【Results】We considered the immunoadsorption as the best method of purifying OECs, whose cells purity achieved 84.66%. But differential anchoring (71.59%) and chemical technique (66.45%) were not desirable because of losing a great deal of dissociated cells; Coating the petri dish was profitable to purify the cells, p75 coating petri dish could not only culture the highest cell purity (88.69%), but also help OECs to anchor rapidly.
【Conclusion】A large number of pured OECs will provide convenience for further basal study and clinical application in future.
【方法】通过对多种方法的比较获得较为理想纯化嗅鞘细胞的措施，嗅球取材直接挤压法，将细胞接种密度统一调整到10~5／ml后应用3种纯化方法各对10份样本处理并作纯度比较；同样密度和接种数量的原代细胞各取10份种植于3种不同的培养皿，对细胞贴壁速度和最终纯度进行检验。最好的纯化嗅鞘细胞方法为免疫吸附法，其细胞纯度达84.66％。而差速贴壁法(71.59％)和化学试剂法(66.45％)均由于损失细胞太多不可取；同时发现包被培养皿对纯化细胞意义重大，P~(75)包被组不仅培养细胞的纯度最高(88.69％)，而且还有利于OECs的快速贴壁。
【结果】我们培养人胚嗅鞘细胞纯度为84.66％，同时发现包被培养皿对纯化细胞意义重大，p75包被不仅培养细胞的纯度高，而且还有利于OECs的快速贴壁。
【结论】此取材及培养方法收获大量嗅鞘细胞将对其进一步的基础研究以及将来应用于临床奠定基础。
【Objective】To develop the better methods of drawing the materials from OB and purifying OECS.
【Methods】We drawing the materials from OB by crushing directly. We had adjusted inoculated density of OECs to uniform 10~5/ml, We made a contrast of cells purity of each 10 samples disposed by three purification methods respectively; The primary cells which possessed the same density and the same inoculating quantity was fetched 10 portions respectively to implant in three kinds of Petri dish. We checked out cellular anchoring velocity and final purity.【Results】We considered the immunoadsorption as the best method of purifying OECs, whose cells purity achieved 84.66%. But differential anchoring (71.59%) and chemical technique (66.45%) were not desirable because of losing a great deal of dissociated cells; Coating the petri dish was profitable to purify the cells, p75 coating petri dish could not only culture the highest cell purity (88.69%), but also help OECs to anchor rapidly.
【Conclusion】A large number of pured OECs will provide convenience for further basal study and clinical application in future.
引文
[1]Pascual JI, Gudino-Cabrera G, Insausti R. Spinal implants of olfactory ensheathing cells promote axon regeneration and bladder activity after bilateral lumbosacral drosal rhizotomy in the adult rat[J]J urol 2002;167(3):1522-1526.
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[3]RamÓn-Cueto A, Cordero MI, Santos-Benito FE Functional recovery of paraplegic rats and motor axon regeneration in their spinal coreds by olfactory ensheathing gila.Neuron 2000 25:425-435.
[4]Imaizumi T, Lankford K L, Kocsis JD. Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord[J].Brain Res .2000,854:70-78.
[5]Ramon-Cueto A, Cordero M I, Santos -Benito F F, et al. Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia [J].Neuron.2000,25:425-435.
[6]Li Y. Field P M, Raisman G Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells[J].Science. 1997,277:2000-2002.
[7]Ramon-Cueto A, Plant G W, Avila J, et al. Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants[J]JNeurosci,1998,18:3803-3815.
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[10] Sonigra RJ, Brighton PC, Jacoby J. Adult rat olfactory nerve ensheathing cell are effective promoters of adult central nervous system neurite outgrowth in coculture.Glia, 1999,25:256-269.
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[24] 杨浩，梁哲等．嗅鞘细胞生物学特性及其在脊髓百生中应用研究动物医学进展，2002，23(6)：29-30．
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[40] 黄红云，王洪美，陈琳等，嗅鞘细胞移植治疗晚期脊髓损伤临床试验初步报告，立体定向和功能性神经外科杂志，2004；17(6)：349～350。
[2]Imaizumi T, Lankford KL, Waxman SG. Transplanted olfactory ensheathing cells remyelinate and enhance axonal conduction in the demyelinated dorsal columns of the rat spinal cord J Neurosci 1998 Aug 15;18(16):6176-6185.
[3]RamÓn-Cueto A, Cordero MI, Santos-Benito FE Functional recovery of paraplegic rats and motor axon regeneration in their spinal coreds by olfactory ensheathing gila.Neuron 2000 25:425-435.
[4]Imaizumi T, Lankford K L, Kocsis JD. Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord[J].Brain Res .2000,854:70-78.
[5]Ramon-Cueto A, Cordero M I, Santos -Benito F F, et al. Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia [J].Neuron.2000,25:425-435.
[6]Li Y. Field P M, Raisman G Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells[J].Science. 1997,277:2000-2002.
[7]Ramon-Cueto A, Plant G W, Avila J, et al. Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants[J]JNeurosci,1998,18:3803-3815.
[8] Ramorr Cueto A, Valverde F. Olfactory bulb ensheathing glia;a unique cell type with axonal growth-promoting properties [J].Glira, 1995,14:163.
[9] Yan H, Nie X, Kocsis JD. Hepatocyte growth factor is a mitogen for olfactory ensheathing cells.J Neurosci Res,2001,66:698-704.
[10] Sonigra RJ, Brighton PC, Jacoby J. Adult rat olfactory nerve ensheathing cell are effective promoters of adult central nervous system neurite outgrowth in coculture.Glia, 1999,25:256-269.
[11] Doucette JR. The glial cells in the nerve fiber layer of the rat olfactory bulb [J] .Anat Rec, 1984,210:385-391.
[12] Ramon-Cueto A, Jesus A. Olfactory ensheathing glia: properties and function[J]. Brain research Bulletin, 1998,46(3): 175-187.
[13] 王琦，周长满，于恩华．成年大鼠嗅球和鼻腔成鞘细胞的粘膜分离培养与鉴定[J]．解剖学报，2002，5(33)：488-491．
[14] 杨浩，游思维，王春婷等．嗅鞘细胞的培养纯化及形态学特征[J]，解剖科学进展，2002，2(8)：102-105．
[15] Ramon-Cueto A, Perez J, Nieto-Sampedro M. In vitro enfolding of olfactory neurites by p75NGF receptor positive ensheathing cells from adult rat olfactory bulb[J].Eur J Neurosci, 1993,5:1172-1180.
[16] Ramorr-Cueto A, Valverde F. Olfactory bulb ensheathing glia: a unique cell type with axonal growth promoting properties[J].Glia, 1995,14:163.
[17] Ramorr-Cueto A, Avila J. Olfactor ensheathing glia: Properties and function[J].Brain Res bull ,1988,46:175.
[18] Gong QZ, Bailey MS, Pixley SK, et al. Localization an deregulation of the low affinity nerve growth factor receptor expression in the rat olfactory system during development and regeneration[J],J Comp Neurol, 1994,344:336.
[19] Rapalino O, Lazarov SO, Agranov E, et al. Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats[J],Nat Med ,1998,4:814.
[20] Li Y, Field PM, Raisman G. Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells[J]. J Neurosci, 1998,18:10514.
[21] 孙天胜，任继鑫，胥少汀．嗅神经鞘细胞桥接脊髓损伤．国外医学[J]．骨科学分册，2002，2(23)：112-114．
[22] Ramon-Cueto A, Plant G W, Avila J, et al. Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheating glia transplants[J].J Neurosci, 1998,18:3803.
[23] Au E, Rokams AJ. Olfactory ensheathing cells of the lamina propria in vivo and in vitro[J].GLIA,2002,41:224-236.
[24] 杨浩，梁哲等．嗅鞘细胞生物学特性及其在脊髓百生中应用研究动物医学进展，2002，23(6)：29-30．
[25] Ramon-Cueto A, Cordero MI, Sanitos FF, et al. Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia[J]. Neuron,2000,25:425-435.
[26] Ramon-Cueto A, Jesus A. Olfactory ensheathing glia:properties and function[J].Brain research Bulletin, 1998,46(3): 175-187.
[27] Wetzer K, Grothe C, Claus E In vitro expression and regulation of ciliary neurotrophic factor and its alpha receptor subunit in neonatal rat olfactory ensheathing cells[J].Neurosci-Lett,2001,306(3): 165-168.
[28] Tisay KT, Key B. The extracellular matrix modulates olfactory neuritis outgrowth on ensheathing cells[J].J-Neurosci,1999,19(22):9890-9899.
[29] Lakatos A, Franklin F, Barnett SC. Olfactory ensheathing cells and Schwann cells differ in their in virto interacions with astrocytes[J]. Glia,2000, 32(3):214-225.
[30] Verdu E, Garcia AG, Fores J, et al. Effects of ensheathign cells transpoanted into photochemically damaged spinal cord[J]. Neuroreport, 2001,12(11):2303-2309.
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