甲基泼尼松龙对体外培养许旺细胞分泌神经营养因子的研究
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摘要
目的:
研究甲基泼尼松龙对体外培养许旺细胞分泌神经营养因子的促进作用。
方法:
两周龄新西兰大白兔6只处死后,用75%酒精浸泡20min,无菌条件下取双侧坐骨神经和臂丛神经,显微镜下仔细剥除神经外膜后,将神经剪成1 mm~3大小,间距1cm种植于预先铺过明胶的培养瓶中;7d后传代,继续培养3d后应用胰酶消化,联合差速贴壁法纯化,并S-100蛋白免疫组化染色检查许旺细胞纯度。鉴定过的许旺细胞配成5×10~4/ml单细胞悬液,每孔1ml加入4孔培养板中。
根据各孔所加入培养液中甲基泼尼松龙浓度的不同进行分组:A孔为对照组,加入含20%小牛血清的双抗高糖DMEM培养液2ml;B孔中加入含0.1mg/L甲基泼尼松龙的DMEM培养液2ml;C孔中加入含1.0mg/L甲基泼尼松龙的DMEM培养液2ml;D孔中加入含5.0mg/L甲基泼尼松龙的DMEM培养液2ml。将培养板置于37℃、CO_2体积分数为5%的恒温培养箱内培养,每天观察细胞生长情况;每2d用随即计数法估算细胞数。每2d吸出培养液分别存于离心管中并4℃冰箱保存;吸取干净后,PBS反复冲洗培养孔,再加入相应培养液。
将前后两次计算的细胞数量分别相减,得出细胞数量的增加值,应用单因素方差分析进行统计学比较。将获得的20管培养液混匀,三等分后,用ELISA法分别做NGF、CNTF、GDNF定量测定,将前后两次的测量值分别相减,得出神经营养因子分泌量的增加值,应用单因素方差分析进行统计学比较。实验数据采用spss13.0统计学软件处理,取P＜0.05为差异有统计学意义。
结果:
①B、C、D组许旺细胞生长速度明显快于A组,三种神经营养因子分泌量明显多于A组,统计学有显著差异(P＜0.05),B、C、D组之间无显著差异(P＞0.05);
②NGF的检测结果有优于CNTF、GDNF的趋势(P＜0.01),CNTF和GDNF的促分泌情况无明显差异(P＞0.05)。
结论:
甲基泼尼松龙能直接作用于许旺细胞来促进体外培养许旺细胞的增殖和神经营养因子的分泌。
甲基泼尼松龙的用量在促进体外培养许旺细胞的增殖和神经营养因子的分泌方面无明显差异。
甲基泼尼松龙能通过促进体外培养许旺细胞的增殖和神经营养因子的分泌来促进损伤周围神经的再生和修复。
Objective:
To research the effects of Methylprednisolone on Schwann cells in vitro of secreting the neurotrophic factor.
Methods:
Chose 6 New Zealand rabbits of 2 weeks-old, killed and soak in ethyl alocohol of 75% for 20 minutes. Procurexing the ischiadic nerve and brachial plexus of both sides under asepsis, striping the epineurium and cutting to be chips of 1 mm3, and then grow in the culture flask which spreading by gelatine with 1 cm distance. Passage after 7days, go on cultivanting 3 days, then trypsinizating and depuration by differential adhesion way. Accredit the purity of cells with S-100 protein-immunohisto-chemistry stain, and preparing to be cell suspension of 5×10~4 /ml, load into 4-hole cultivation board with 1 ml/hole.
According to the concentration of the drug, there was divided into four groups: A group, control group, only giving DMEM- culture fluid 2ml; B group, adding 0.1mg / L methylprednisolone into the culture fluid; C group, adding 1.0mg/L methylprednisolone into the culture fluid; D group, to join 5.0mg/L methylprednisolone into the culture fluid. Cultivate in the homeothermia incubator of 37℃with 20% CO_2. Observe the cell growth and estimate the cell number. Every two days, draw-off the culture fluid and keep in the refrigerator of 4℃, wash the board by PBS, then join the new culture fluid with different group.
Calculate the increase value of the cell number, get the quantitative assay of NGF、CNTF、GDNF by ELISA, and Calculate the increase value. The statistics test was carried out with one-side variance analysis by SPSS 13.0, choosing statistical significance of P<0.05.
Results:
①B, C, D groups Schwann cells faster than the growth rate of Group A, three kinds of neurotrophic factor secreted significantly more than group A, statistically significant differences (P <0.01), B, C, D Group Between the secretion was no significant difference between the number of (P> 0.05);
②NGF of the test results are better than that of CNTF, GDNF trend (P <0.01), CNTF and GDNF promote the secretion of the situation there was no significant difference (P> 0.05).
Conclusion:
Methylprednisolone can promote the proliferation of Schwann cells in vitro and the secretion of neurotrophic factor. Application of low-dose and high-dose has no significant differences.
Methylprednisolone can promote the regenerate and repair of peripheral nerve by promoting the proliferation of Schwann cells and the secretion of neurotrophic factor.
研究甲基泼尼松龙对体外培养许旺细胞分泌神经营养因子的促进作用。
方法:
两周龄新西兰大白兔6只处死后,用75%酒精浸泡20min,无菌条件下取双侧坐骨神经和臂丛神经,显微镜下仔细剥除神经外膜后,将神经剪成1 mm~3大小,间距1cm种植于预先铺过明胶的培养瓶中;7d后传代,继续培养3d后应用胰酶消化,联合差速贴壁法纯化,并S-100蛋白免疫组化染色检查许旺细胞纯度。鉴定过的许旺细胞配成5×10~4/ml单细胞悬液,每孔1ml加入4孔培养板中。
根据各孔所加入培养液中甲基泼尼松龙浓度的不同进行分组:A孔为对照组,加入含20%小牛血清的双抗高糖DMEM培养液2ml;B孔中加入含0.1mg/L甲基泼尼松龙的DMEM培养液2ml;C孔中加入含1.0mg/L甲基泼尼松龙的DMEM培养液2ml;D孔中加入含5.0mg/L甲基泼尼松龙的DMEM培养液2ml。将培养板置于37℃、CO_2体积分数为5%的恒温培养箱内培养,每天观察细胞生长情况;每2d用随即计数法估算细胞数。每2d吸出培养液分别存于离心管中并4℃冰箱保存;吸取干净后,PBS反复冲洗培养孔,再加入相应培养液。
将前后两次计算的细胞数量分别相减,得出细胞数量的增加值,应用单因素方差分析进行统计学比较。将获得的20管培养液混匀,三等分后,用ELISA法分别做NGF、CNTF、GDNF定量测定,将前后两次的测量值分别相减,得出神经营养因子分泌量的增加值,应用单因素方差分析进行统计学比较。实验数据采用spss13.0统计学软件处理,取P＜0.05为差异有统计学意义。
结果:
①B、C、D组许旺细胞生长速度明显快于A组,三种神经营养因子分泌量明显多于A组,统计学有显著差异(P＜0.05),B、C、D组之间无显著差异(P＞0.05);
②NGF的检测结果有优于CNTF、GDNF的趋势(P＜0.01),CNTF和GDNF的促分泌情况无明显差异(P＞0.05)。
结论:
甲基泼尼松龙能直接作用于许旺细胞来促进体外培养许旺细胞的增殖和神经营养因子的分泌。
甲基泼尼松龙的用量在促进体外培养许旺细胞的增殖和神经营养因子的分泌方面无明显差异。
甲基泼尼松龙能通过促进体外培养许旺细胞的增殖和神经营养因子的分泌来促进损伤周围神经的再生和修复。
Objective:
To research the effects of Methylprednisolone on Schwann cells in vitro of secreting the neurotrophic factor.
Methods:
Chose 6 New Zealand rabbits of 2 weeks-old, killed and soak in ethyl alocohol of 75% for 20 minutes. Procurexing the ischiadic nerve and brachial plexus of both sides under asepsis, striping the epineurium and cutting to be chips of 1 mm3, and then grow in the culture flask which spreading by gelatine with 1 cm distance. Passage after 7days, go on cultivanting 3 days, then trypsinizating and depuration by differential adhesion way. Accredit the purity of cells with S-100 protein-immunohisto-chemistry stain, and preparing to be cell suspension of 5×10~4 /ml, load into 4-hole cultivation board with 1 ml/hole.
According to the concentration of the drug, there was divided into four groups: A group, control group, only giving DMEM- culture fluid 2ml; B group, adding 0.1mg / L methylprednisolone into the culture fluid; C group, adding 1.0mg/L methylprednisolone into the culture fluid; D group, to join 5.0mg/L methylprednisolone into the culture fluid. Cultivate in the homeothermia incubator of 37℃with 20% CO_2. Observe the cell growth and estimate the cell number. Every two days, draw-off the culture fluid and keep in the refrigerator of 4℃, wash the board by PBS, then join the new culture fluid with different group.
Calculate the increase value of the cell number, get the quantitative assay of NGF、CNTF、GDNF by ELISA, and Calculate the increase value. The statistics test was carried out with one-side variance analysis by SPSS 13.0, choosing statistical significance of P<0.05.
Results:
①B, C, D groups Schwann cells faster than the growth rate of Group A, three kinds of neurotrophic factor secreted significantly more than group A, statistically significant differences (P <0.01), B, C, D Group Between the secretion was no significant difference between the number of (P> 0.05);
②NGF of the test results are better than that of CNTF, GDNF trend (P <0.01), CNTF and GDNF promote the secretion of the situation there was no significant difference (P> 0.05).
Conclusion:
Methylprednisolone can promote the proliferation of Schwann cells in vitro and the secretion of neurotrophic factor. Application of low-dose and high-dose has no significant differences.
Methylprednisolone can promote the regenerate and repair of peripheral nerve by promoting the proliferation of Schwann cells and the secretion of neurotrophic factor.
引文
1.Frostick SP,Yin Q,Kemp GJ.Schwann cells,neurotrophic factors,and peripheral nerve regeneration[J].Microsurgery,1998,18:397-402.
2.甘思德,范明.神经退变和再生的构筑变化[J].中国应用生理学杂志,1993,9:322-330.
3.Jessen KR,Mirsky R.Embryonic Schwann cells development:the biology of Schwann cells precursors and early Schwann cells.J Anat,1997,191:501-505.
4.张继东,李国栋,赵定麟.甲基强的松龙治疗脊髓损伤的机制[J].颈腰痛杂志,1997,18:207-209.
5.Baffour R,Achanta K,Kaufman J,et al.Synergistic effect of basic fibroblast growth factor and methylprednisolone on neurological function after exoerimental spinal cord injury[J].J Neurosurg,1995,83:105-110.
6.姜保国,党育,张殿英等.甲基泼尼松龙对周围神经修复的影响[J].中华外科杂志,2001,39:476-479.
7.党育,姜保国,张殿英等.甲基泼尼松龙局部应用对周围神经修复的影响.中华实验外科杂志[J],2002,19(增刊):139-140.
8.党育,姜保国,张殿英等.甲基泼尼松龙局部应用对周围神经损伤修复后的形态学研究[J].中华手外科杂志,2005,10:314-316.
9.蔡鸿敏,吴学建.甲基泼尼松龙对体外培养兔雪旺细胞迁移及增殖的影响[J].郑州大学学报(医学版),2008,43:312
10.Yin Q,Kemp G J,Frostick SP.Neurotrophins,neurones and peripheral nerve regeneration.J Hand Surg,1998,23:433-437.
11.Wheeler EF,Naftel JP,Pan M,et al.Neurotrophin receptor expression is induced in a subpopulation of trigeminal neurons that label by retrograde transport of NGF or fluoro-gold following tooth injury[J].Brain Res Mol Brain Res,1998,61:23-38.
12.Frostick SP,Yin Q,Kemp GJ.Schwann cells,neurotrophic factors,and peripheral nerve regeneration[J].Microsurgery,1998,18:397-405.
13.程飚,陈峥嵘.成年兔坐骨神经雪旺细胞的分离纯化培养[J].复旦学报(医学科学版),2001,28:230-232.
14.翁惠兰,孙淑明等.雪旺细胞体外培养和纯化的新方法[J].中国基层医药,2005,12:1299-1301.
15.刘婷;李世普等.体外原代培养成年大鼠许旺细胞的实验方法:组织块反复种植+低浓度酶消化+差速贴壁[J].中国组织工程研究与临床康复,2007,11:5545-5547.
16.何继银,劳杰.周围神经损伤后雪旺细胞生存与凋亡的动态平衡[J].中华创伤骨科杂志,2004,6:330.
17.Peter E,Gabriel,John Y,et al.Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor[J].Neurosci,1999,19:1708-1716.
18.Sulaiman OA,Gordon T.Effects of short-and-long-term Schwann cell denervation on peripheral nerve regeneration,myelination,and size[J].Glia,2000,32:234-246.
19.Torigoe K.The role of migratory Schwann cells in nerve regeneration as studied by the film model[J].J Peripher Nerv Syst,1997,2:227-231.
20.Lindholm D,Heumann R,MeyerM,et al.Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve[J].Nature,1987,330:658-659.
21.Yin Y,Cui Q,Li Y,et al.Macrophage-Derived Factors Stimulate Optic Nerve Regeneration[J].J Neurosci,2003,23:2284-2293.
22.Singh N,Birdi TJ,Antia NU.Differential in vitro modulation of Schwann cell proliferation by Mycobacterium leprae and macrophages in the murine strains,Swiss white and C57B1/6[J].J Peripher Nerv Syst,1998,3:207-216.
23.Ramesh V.Merlin and the ERM proteins in Schwann cells,neurons and growth cones[J].Nat Rev Neurosci,2004,5:462-470.
24.Dubey N,Letourneau PC,Tranquillo RT.Guided neurite elongation and Schwann cell invasion into magnetically aligned collagen in simulated peripheral nerve regeneration[J].Exp Neurol,1999,158:338-350.
25.王国英,平井圭一,钟世镇等.雪旺细胞基底膜成分LN在神经再生的作用探讨[J].中国临床解剖学杂志,1992,10:200-207.
26.Dezawa M,Nagano T.Immunohistochemical localization of cell adhesion molecules and cell-cell contact proteins during regeneration of the rat optic nerve induced by sciatic nerve autotransplantation[J].Anat Rce,1996,246:114-126.
27.Dubovy P.Schwann cells and endoneurial extracellular matrix molecules as potential cues for sorting of regenerated axons[J].Anat Sci Int,2004,79:198-208.
28.Dezawa M,Mutoh T,Dezawa A,et al.Tight junctions between the axon and Schwann cell during PNS regeneration[J].Neuroreport,1996,7:1829-1832.
29.Dezawa M,Mutoh T,Dezawa A,et al.Putative gap junctional communication between axon and regenerating Schwann cells during mammalian peripheral nerve regeneration.Neuroscience,1998,85:663-667.
30.Aguayo AJ,Epps J,Charron L,et al.Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves:quantitative microscopy and radioautography[J].Brain Res,1976,104:1-20.
31.Semkova I,Krieglstein J.Neuroprotection mediated via neurotrophic factors and induction of neurotrophic factors[J].Brain Res Brain Res Rev,1999,30:176-188.
32.Salka T,Senhe E,Noguchi I,et al.Effects of nerve crush and transection on mRNA levels for growth factor in the facial motoneurons[J].Molec Brain Res,1991,9:157.
33.Newman JP,Verity AN,Hawatmeh S,et al.Ciliary neurotrophic factor enhanace peripheral nerve regeneration[J].Arch Otolaryngol Head Neck Surg,1996,122:399-403.
34.Yoon DH,Kim YS,Young W.Therapeutic time window for methylprednisolone in spinal cord injured rat[J].Yonsei Med J.1999,40:313-320.
35.Ildan F,Oner A,Polat S,et aI.Correlation of alterations on Na(+)-K+/Mg+2ATPase activity,lipid peroxidation and oltrastructural findings following experimental spinal cord injury with and without intravenous methylprednisolone treatment.Neurosurg Rev,1995,18:35-44.
36.张继东,李国栋,赵定麟.甲基强的松龙治疗脊髓损伤的机制[J].颈腰痛杂志,1997,18:207-209.
37.Baffour R,Achanta K,Kaufman J,et al.Synergistic effect of basic fibroblast growth factor and methylprednisolone on neurological function after exoerimental spinal cord injury[J].J Neurosurg,1995,83:105-110.
38.杜蝉,姜保国.许旺细胞增殖和分化过程中的信号通路[J].中国组织工程研究与临床康复,2007,11:563-565.
39.李平,许扬滨等.许旺细胞源神经营养因子在运动神经元轴突逆行运输的研究[J].中华显微外科杂志,2002,25:278-279.
40.Garret AN,Britsch S,Birchmeiet C.Neuregulin,a factor with many functions in the life of a schwann cell.Bioessays 2000;22:987-990.
41.李皓桓,刘世清等.吡咯喹啉醌对许旺细胞合成与分泌神经生长因子的影响[J].中华显微外科杂志,2005,28:242-245.
1.Jessen KR,Mirsky R.Embryonic Schwann cells development:the biology of Schwann cells precursors and early Schwann cells[J].J Anat,1997,191:501-505.
2.甘思德,范明.神经退变和再生的构筑变化[J].中国应用生理学杂志.1993,9:322-324.
3.Torigoe K.The role of migratory Schwann cells in nerve regeneration as studied by the film model[J].J Peripher Nerv Syst,1997,2:227-231.
4.Kunjor T,Hirohito FT,Akira T,et al.Basic behavior of migritory Schwann cells in peripheral nerve regeneration[J].Exp Neuro,1996,137:301-304.
5.陈国奋,顾立强,裴国献.免疫抑制状态下的周围神经再生[J].中国修复重建外科杂志.2002,16:139-141.
6.Ansselin AD,Pollard JD.Immunopathological factors in peripheral nerve allograft rejection:quantification of lymphocyte invasion and major histocompatibility complex expression[J].Neurol Sci,1990,96:75-81.
7.裴福兴,杨志明,黄富国等.周围神经损伤后的免疫反应和神经再生[J].中华显微外科杂志,1995;18:146-151.
8.Mitchell GW,Williams GS,Bosch EP.Class Ⅱ antigen expression in peripheral neuropathies[J].J Neurol Sci,1991,102:170-175.
9.Scarpini E,Lisak RP,Beretta S,et al.Quantitative assessment of classⅡMoleculars in normal and pathological nerves:Immunocytochemical studies in vivo and in tissue culture.Brain,1990;113:659-662.
10.王光林,裴福兴,杨志明.周围神经损伤后HLA-DR抗原的表达[J].中华手外科杂志.1996;12:170-172.
11.Mackinnon SE,Kelly L,Hunter DA.Comparison of regeneration across a vascularized versus conventional nervegraft[J].Microsurgery,1988;9:226-230.
12.Midha R,Mackinnon SE,Evans PJ,et al.Comparison of regeneration across nerve allografts with temporary or continuous Cyclosporine an immunosuppression.[J]Neurosurg,1993,13:92-94.
13.Bain JR,Mackinnon SE,Hudson AR,et al.The peripheral Nerveallo graft:An assessment of regeneration across nerve Allograft in rats immunosuppressed with cyclosporine A.Plast Reconstr Surg,1988,82:1052-1061.
14.Buttemeyer R,Rao U,Jones NF.Peripheral nerve allograft transplantation with FK506:functional,histological,and immunological results before and after discontinuation ofimmunosuppression.Ann Plastic Surg,1995,35:396-399.
15.Nakao Y,Mackinnon SE,Mohanakumar T,et al.Monoclonal antibodies against ICAM-1 and LFA-1(CD11A) induce specific tolerance to peripheral nerve allograft in rats.Transplant Proc,1995,27:373-375.
16.Matsuyama T,Mackay M,Midha R.Peripheral nerve repair and grafting techniques:a review[J].Neurol Med Chir(Tokyo),2000,40:187-199.
17.Machinnon SE.A study of nerve regeneration across synthetic nerve conduit for gap up to 5 cm in theprimate[J].J Reconstr Microsurg,1990,16:117-129.
18.Glasby MA.Specifity for homonymous pathways following repair of peripheral nerves with treated skeletal muscle autografts in the primate[J].Br J Plast Surg,1991,44:135-143.
19.苟三怀,侯春林.几丁质桥接周围神经缺损的实验研究[J].中国修复重建外科杂志,1993,7:169-172.
20.王碧波,戴传昌.周围神经缺损的组织工程修复进展[J].组织工程与重建外科杂志,2006,2:113-115.
21.nerve regeneration through combined skeletal muscle and vein conduit grafts[J].Microsurgery,2000,20:65-71.
22.Battiston B,Tos P,Cushway TR.Nerve repair by means of vein filled with muscle grafts Ⅰ[J].Clinical results.Microsurgery,2000,20:32-36.
23.Seniuk NA.Neurotrophic factors:a role in peripheral neuron survival and axonal repair[J].J Reconstr Microsurg,1992,8:399-404.
24.Spector JG,Lee P,Derby A,et al.Rabbit facial nerve regeneration in NGF-containing silastic tubes[J].Laryngoscope,1993,103:548-556.
25.Utley DS,Lewin SL,Cheng ET,et al.Brain-derived neurotrophic factor and collagen tubulization enhance functional recovery after peripheral nerve transection and repair[J].Arch Otolaryngol Head Neck Surg,1996,122:407-413.
26.Sorensen J,Hase G,Krarup C,et al.Gene transfer to Schwann cells after peripheral nerve injury:a delivery system for therapeutic agents[J].Ann Neurol,1998,43:205-211.
27.Gravel C,Gotz R,Lorrain A,et al.Adenoviral gene transfer of ciliary neurotrophic factor and brain-derived neurotrophic factor leads to longterm survival ofaxotomized motor neurons[J].Nat Med,1997,3:765-770.
28.李金照,陈燕,夏另朝,等.脑源神经营养因子基因转染神经断端促进切断坐骨神经再生[J].生物物理学报,1997,13:448-452.
29.Watabe K,Ohashi T,Sakamoto T,et al.Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of gliaI cell line-derived neurotrophic facto[J].J Neurosci Res,2000,6:511-519.
30.Peter E,Gabriel,John Y,et al.Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor[J].Neurosci,1999,19:1708-1716.
31.Sulaiman OA,Gordon T.Effects of short-and-long-term Schwann cell denervation on peripheral nerve regeneration,myelination,and size[J].Glia,2000,32:234-246.
32.Garratt AN,Voiculescu O,Topilko P,et al.A dual role of erbB2 in myelination and in expansion of the Schwann cell precursor pool.J Cell Biol,2000,148:1035-1046.
33.Ahmed Z,Underwood S,Brown RA.Low concentrations of fibrinogen increase cell migration speed on fibronectin/fibrinogen composite cables.Cell Motil Cytoskeleton,2000,46:6-16.
34.Torigoe K,Awaya A.A newly synthesized neurotropic pyrimidine compound,MS-818,may activate migratory Schwann cells in peripheral nerve regeneration.Brain Res,1998,787:337-340.
35.李强,李民,伍亚民.物理因子影响周围神经再生的研究概况.中华物理医学与康复杂志,2003,25:186-187.
36.Dubey N,Letoumeau PC,Tranquillo RT.Guided neurite elongation and Schwann cell invasion into magnetically aligned collagen in simulated peripheral nerve regeneration.Exp Neurol,1999,158:338-350.
37.Yin Q,Kemp GJ,Frostick SP.Neurotrophins,neurones and peripheral nerve regeneration.J Hand Surg,1998,23:433-437.
38.Wheeler EF,Naftel JP,Pan M,et al.Neurotrophin receptor expression is induced in a subpopulation of trigeminal neurons that label by retrograde transport of NGF or fluoro-gold following tooth injury[J].Brain Res Mol Brain Res,1998,61:23-38.
39.Frostick SP,Yin Q,Kemp GJ.Schwann cells,neurotrophic factors,and peripheral nerve regeneration[J].Microsurgery,1998,18:397-405.
40.Semkova I,Krieglstein J.Neuroprotection mediated via neurotrophic factors and induction of neurotrophic factors[J].Brain Res Brain Res Rev,1999,30:176-188.
41.Salka T,Senhe E,Noguchi I,et al.Effects of nerve crush and transection on mRNA levels for growth factor in the facial motoneurons[J].Molec Brain Res,1991,9:157.
42.Newman JP,Verity AN,Hawatmeh S,et al.Ciliary neurotrophic factor enhanace peripheral nerve regeneration.Arch Otolaryngol Head Neck Surg,1996,122:399403.
43.Inoue T,Hosokawa M,Morigiwa K,et al.Bcl-2 overexpression does not enhance in vivo axonal regeneration of retinal ganglion cells after peripheral nerve transplantation in adult mice[J].J Neurosci,2002,22:4468-4477.
44.Hammarberg H.Piehl F.Cullheim S,et al.GDNF mRNA in Schwann cells and DRG satellite ceil after chronic sciatic nerve injury.Neuroreport,1996,7:857-860.
45.Ronatd WO,Lucien J,James E,et al.Developing motor neurons rescued from programmed and axotomy-induced cell death by glial cell line-derived neurotrophic factor.Nature,1995,373:344.
46.Grothe C,Meisinger C,Herrenstein A,et aL.Expression of fibroblast growth factor-2 and fibroblast growth factor-1 messenger RNAs in Spinal ganglia and sciatic nerve:regeneration after peripheral nerve lesion.Neurosci,1996,76:123-35.
47.王非,翁龙江,李主一.碱性成纤维细胞生长因子促进神经再生的研究进展.中华显微外科杂志,1999,22:76.
48.Dezawa M,Adachi-Usami E.Role of Schwann cells in retinal ganglion cell axon regeneration[J].Prog Retin Eye Res,2000,19:171-204.
49.Lohof AM,Lp nY,Poo MM,et al.Potentiation of developing neuromuscular synapses by neurotrophins NT-3 and BDNF[J].Nature,1993,363:350-353.
50.David BP,Ambily B.Krox-20 inhibits Jun-NH2-terminal kinase/c-Jun to control Schwann cell proliferation and death[J].J CellBiol,2004,164:385-394.
51.朱浩,丁文龙.施旺细胞发育及其调控的信号通路[J].解剖科学进展,2006,12:376-380.
52.Frohnert PW,StonecypherMS,Carroll SL.Constitutive activation of the neuregulin-1 /erbB receptor signaling pathway is essential for the proliferation of a neoplastic Schwann cell line[J].Glia,2003,43:104-118.
53.Fallon KB,Havlioglu N,Hamilton LH,et al.Constitutive activation of the neuregulin-1/erbB signaling pathway promotes the proliferation of a human peripheral neuroepithelioma cell line[J].J Neurooncol,2004,66:273-284.
54.张继东,候铁胜,赵定麟.太剂量甲基强的松龙对大鼠损伤脊髓中神经生长因子的影响[J].中华外科杂志,1995,12:727.
55.Nachemson AK,Lundborg G,Myrhage R,et al.Nerve regeneration and pharmacological suppression of the scar reaction at the suture site.An experimental study on the effect of estrogen-progesterone,methylprednisoloneacetate and cis-hydroxyproline in rat sciatic nerve.Scand J Plast Reconstr Surg,1985,19:255-260.
56.姜保国,党育,张殿英等.甲基泼尼松龙对周围神经修复的影响[J].中华外科 杂志,2001,39:476-479.
57.党育,姜保国,张殿英等.甲基泼尼松龙局部应用对周围神经修复的影响[J].中华实验外科杂志,2002,19(增刊):139-140.
58.党育,姜保国,张殿英等.甲基泼尼松龙局部应用对周围神经损伤修复后的形态学研究[J].中华手外科杂志,2005,10:314-316.
59.蔡鸿敏,吴学建.甲基泼尼松龙对体外培养兔雪旺细胞迁移及增殖的影响[J].郑州大学学报(医学版),2008,43:312-314.
60.万虹,孙梅珍.雪旺氏细胞培养方法的比较[J].中国危重病急救医学2001,13(9):530-532.
61.翁惠兰,孙淑明等.雪旺细胞体外培养和纯化的新方法[J].中国基层医药,2005,12:1299-1302.
62.Friedenstein AJ.Precursor cells of mesenchymal[J].Int Rev Cytol 1976,47:327-55.
63.张培训,姜保国.骨髓间充质干细胞的研究进展与周围神经修复[J].中国临床康复杂志,2004,8:326-7.
64.吴志伟,陈峥嵘等.人胚雪旺细胞组织工程神经修复坐骨神经缺损的实验研究[J].中国临床医学,2006,13:810-3.
2.甘思德,范明.神经退变和再生的构筑变化[J].中国应用生理学杂志,1993,9:322-330.
3.Jessen KR,Mirsky R.Embryonic Schwann cells development:the biology of Schwann cells precursors and early Schwann cells.J Anat,1997,191:501-505.
4.张继东,李国栋,赵定麟.甲基强的松龙治疗脊髓损伤的机制[J].颈腰痛杂志,1997,18:207-209.
5.Baffour R,Achanta K,Kaufman J,et al.Synergistic effect of basic fibroblast growth factor and methylprednisolone on neurological function after exoerimental spinal cord injury[J].J Neurosurg,1995,83:105-110.
6.姜保国,党育,张殿英等.甲基泼尼松龙对周围神经修复的影响[J].中华外科杂志,2001,39:476-479.
7.党育,姜保国,张殿英等.甲基泼尼松龙局部应用对周围神经修复的影响.中华实验外科杂志[J],2002,19(增刊):139-140.
8.党育,姜保国,张殿英等.甲基泼尼松龙局部应用对周围神经损伤修复后的形态学研究[J].中华手外科杂志,2005,10:314-316.
9.蔡鸿敏,吴学建.甲基泼尼松龙对体外培养兔雪旺细胞迁移及增殖的影响[J].郑州大学学报(医学版),2008,43:312
10.Yin Q,Kemp G J,Frostick SP.Neurotrophins,neurones and peripheral nerve regeneration.J Hand Surg,1998,23:433-437.
11.Wheeler EF,Naftel JP,Pan M,et al.Neurotrophin receptor expression is induced in a subpopulation of trigeminal neurons that label by retrograde transport of NGF or fluoro-gold following tooth injury[J].Brain Res Mol Brain Res,1998,61:23-38.
12.Frostick SP,Yin Q,Kemp GJ.Schwann cells,neurotrophic factors,and peripheral nerve regeneration[J].Microsurgery,1998,18:397-405.
13.程飚,陈峥嵘.成年兔坐骨神经雪旺细胞的分离纯化培养[J].复旦学报(医学科学版),2001,28:230-232.
14.翁惠兰,孙淑明等.雪旺细胞体外培养和纯化的新方法[J].中国基层医药,2005,12:1299-1301.
15.刘婷;李世普等.体外原代培养成年大鼠许旺细胞的实验方法:组织块反复种植+低浓度酶消化+差速贴壁[J].中国组织工程研究与临床康复,2007,11:5545-5547.
16.何继银,劳杰.周围神经损伤后雪旺细胞生存与凋亡的动态平衡[J].中华创伤骨科杂志,2004,6:330.
17.Peter E,Gabriel,John Y,et al.Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor[J].Neurosci,1999,19:1708-1716.
18.Sulaiman OA,Gordon T.Effects of short-and-long-term Schwann cell denervation on peripheral nerve regeneration,myelination,and size[J].Glia,2000,32:234-246.
19.Torigoe K.The role of migratory Schwann cells in nerve regeneration as studied by the film model[J].J Peripher Nerv Syst,1997,2:227-231.
20.Lindholm D,Heumann R,MeyerM,et al.Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve[J].Nature,1987,330:658-659.
21.Yin Y,Cui Q,Li Y,et al.Macrophage-Derived Factors Stimulate Optic Nerve Regeneration[J].J Neurosci,2003,23:2284-2293.
22.Singh N,Birdi TJ,Antia NU.Differential in vitro modulation of Schwann cell proliferation by Mycobacterium leprae and macrophages in the murine strains,Swiss white and C57B1/6[J].J Peripher Nerv Syst,1998,3:207-216.
23.Ramesh V.Merlin and the ERM proteins in Schwann cells,neurons and growth cones[J].Nat Rev Neurosci,2004,5:462-470.
24.Dubey N,Letourneau PC,Tranquillo RT.Guided neurite elongation and Schwann cell invasion into magnetically aligned collagen in simulated peripheral nerve regeneration[J].Exp Neurol,1999,158:338-350.
25.王国英,平井圭一,钟世镇等.雪旺细胞基底膜成分LN在神经再生的作用探讨[J].中国临床解剖学杂志,1992,10:200-207.
26.Dezawa M,Nagano T.Immunohistochemical localization of cell adhesion molecules and cell-cell contact proteins during regeneration of the rat optic nerve induced by sciatic nerve autotransplantation[J].Anat Rce,1996,246:114-126.
27.Dubovy P.Schwann cells and endoneurial extracellular matrix molecules as potential cues for sorting of regenerated axons[J].Anat Sci Int,2004,79:198-208.
28.Dezawa M,Mutoh T,Dezawa A,et al.Tight junctions between the axon and Schwann cell during PNS regeneration[J].Neuroreport,1996,7:1829-1832.
29.Dezawa M,Mutoh T,Dezawa A,et al.Putative gap junctional communication between axon and regenerating Schwann cells during mammalian peripheral nerve regeneration.Neuroscience,1998,85:663-667.
30.Aguayo AJ,Epps J,Charron L,et al.Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves:quantitative microscopy and radioautography[J].Brain Res,1976,104:1-20.
31.Semkova I,Krieglstein J.Neuroprotection mediated via neurotrophic factors and induction of neurotrophic factors[J].Brain Res Brain Res Rev,1999,30:176-188.
32.Salka T,Senhe E,Noguchi I,et al.Effects of nerve crush and transection on mRNA levels for growth factor in the facial motoneurons[J].Molec Brain Res,1991,9:157.
33.Newman JP,Verity AN,Hawatmeh S,et al.Ciliary neurotrophic factor enhanace peripheral nerve regeneration[J].Arch Otolaryngol Head Neck Surg,1996,122:399-403.
34.Yoon DH,Kim YS,Young W.Therapeutic time window for methylprednisolone in spinal cord injured rat[J].Yonsei Med J.1999,40:313-320.
35.Ildan F,Oner A,Polat S,et aI.Correlation of alterations on Na(+)-K+/Mg+2ATPase activity,lipid peroxidation and oltrastructural findings following experimental spinal cord injury with and without intravenous methylprednisolone treatment.Neurosurg Rev,1995,18:35-44.
36.张继东,李国栋,赵定麟.甲基强的松龙治疗脊髓损伤的机制[J].颈腰痛杂志,1997,18:207-209.
37.Baffour R,Achanta K,Kaufman J,et al.Synergistic effect of basic fibroblast growth factor and methylprednisolone on neurological function after exoerimental spinal cord injury[J].J Neurosurg,1995,83:105-110.
38.杜蝉,姜保国.许旺细胞增殖和分化过程中的信号通路[J].中国组织工程研究与临床康复,2007,11:563-565.
39.李平,许扬滨等.许旺细胞源神经营养因子在运动神经元轴突逆行运输的研究[J].中华显微外科杂志,2002,25:278-279.
40.Garret AN,Britsch S,Birchmeiet C.Neuregulin,a factor with many functions in the life of a schwann cell.Bioessays 2000;22:987-990.
41.李皓桓,刘世清等.吡咯喹啉醌对许旺细胞合成与分泌神经生长因子的影响[J].中华显微外科杂志,2005,28:242-245.
1.Jessen KR,Mirsky R.Embryonic Schwann cells development:the biology of Schwann cells precursors and early Schwann cells[J].J Anat,1997,191:501-505.
2.甘思德,范明.神经退变和再生的构筑变化[J].中国应用生理学杂志.1993,9:322-324.
3.Torigoe K.The role of migratory Schwann cells in nerve regeneration as studied by the film model[J].J Peripher Nerv Syst,1997,2:227-231.
4.Kunjor T,Hirohito FT,Akira T,et al.Basic behavior of migritory Schwann cells in peripheral nerve regeneration[J].Exp Neuro,1996,137:301-304.
5.陈国奋,顾立强,裴国献.免疫抑制状态下的周围神经再生[J].中国修复重建外科杂志.2002,16:139-141.
6.Ansselin AD,Pollard JD.Immunopathological factors in peripheral nerve allograft rejection:quantification of lymphocyte invasion and major histocompatibility complex expression[J].Neurol Sci,1990,96:75-81.
7.裴福兴,杨志明,黄富国等.周围神经损伤后的免疫反应和神经再生[J].中华显微外科杂志,1995;18:146-151.
8.Mitchell GW,Williams GS,Bosch EP.Class Ⅱ antigen expression in peripheral neuropathies[J].J Neurol Sci,1991,102:170-175.
9.Scarpini E,Lisak RP,Beretta S,et al.Quantitative assessment of classⅡMoleculars in normal and pathological nerves:Immunocytochemical studies in vivo and in tissue culture.Brain,1990;113:659-662.
10.王光林,裴福兴,杨志明.周围神经损伤后HLA-DR抗原的表达[J].中华手外科杂志.1996;12:170-172.
11.Mackinnon SE,Kelly L,Hunter DA.Comparison of regeneration across a vascularized versus conventional nervegraft[J].Microsurgery,1988;9:226-230.
12.Midha R,Mackinnon SE,Evans PJ,et al.Comparison of regeneration across nerve allografts with temporary or continuous Cyclosporine an immunosuppression.[J]Neurosurg,1993,13:92-94.
13.Bain JR,Mackinnon SE,Hudson AR,et al.The peripheral Nerveallo graft:An assessment of regeneration across nerve Allograft in rats immunosuppressed with cyclosporine A.Plast Reconstr Surg,1988,82:1052-1061.
14.Buttemeyer R,Rao U,Jones NF.Peripheral nerve allograft transplantation with FK506:functional,histological,and immunological results before and after discontinuation ofimmunosuppression.Ann Plastic Surg,1995,35:396-399.
15.Nakao Y,Mackinnon SE,Mohanakumar T,et al.Monoclonal antibodies against ICAM-1 and LFA-1(CD11A) induce specific tolerance to peripheral nerve allograft in rats.Transplant Proc,1995,27:373-375.
16.Matsuyama T,Mackay M,Midha R.Peripheral nerve repair and grafting techniques:a review[J].Neurol Med Chir(Tokyo),2000,40:187-199.
17.Machinnon SE.A study of nerve regeneration across synthetic nerve conduit for gap up to 5 cm in theprimate[J].J Reconstr Microsurg,1990,16:117-129.
18.Glasby MA.Specifity for homonymous pathways following repair of peripheral nerves with treated skeletal muscle autografts in the primate[J].Br J Plast Surg,1991,44:135-143.
19.苟三怀,侯春林.几丁质桥接周围神经缺损的实验研究[J].中国修复重建外科杂志,1993,7:169-172.
20.王碧波,戴传昌.周围神经缺损的组织工程修复进展[J].组织工程与重建外科杂志,2006,2:113-115.
21.nerve regeneration through combined skeletal muscle and vein conduit grafts[J].Microsurgery,2000,20:65-71.
22.Battiston B,Tos P,Cushway TR.Nerve repair by means of vein filled with muscle grafts Ⅰ[J].Clinical results.Microsurgery,2000,20:32-36.
23.Seniuk NA.Neurotrophic factors:a role in peripheral neuron survival and axonal repair[J].J Reconstr Microsurg,1992,8:399-404.
24.Spector JG,Lee P,Derby A,et al.Rabbit facial nerve regeneration in NGF-containing silastic tubes[J].Laryngoscope,1993,103:548-556.
25.Utley DS,Lewin SL,Cheng ET,et al.Brain-derived neurotrophic factor and collagen tubulization enhance functional recovery after peripheral nerve transection and repair[J].Arch Otolaryngol Head Neck Surg,1996,122:407-413.
26.Sorensen J,Hase G,Krarup C,et al.Gene transfer to Schwann cells after peripheral nerve injury:a delivery system for therapeutic agents[J].Ann Neurol,1998,43:205-211.
27.Gravel C,Gotz R,Lorrain A,et al.Adenoviral gene transfer of ciliary neurotrophic factor and brain-derived neurotrophic factor leads to longterm survival ofaxotomized motor neurons[J].Nat Med,1997,3:765-770.
28.李金照,陈燕,夏另朝,等.脑源神经营养因子基因转染神经断端促进切断坐骨神经再生[J].生物物理学报,1997,13:448-452.
29.Watabe K,Ohashi T,Sakamoto T,et al.Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of gliaI cell line-derived neurotrophic facto[J].J Neurosci Res,2000,6:511-519.
30.Peter E,Gabriel,John Y,et al.Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor[J].Neurosci,1999,19:1708-1716.
31.Sulaiman OA,Gordon T.Effects of short-and-long-term Schwann cell denervation on peripheral nerve regeneration,myelination,and size[J].Glia,2000,32:234-246.
32.Garratt AN,Voiculescu O,Topilko P,et al.A dual role of erbB2 in myelination and in expansion of the Schwann cell precursor pool.J Cell Biol,2000,148:1035-1046.
33.Ahmed Z,Underwood S,Brown RA.Low concentrations of fibrinogen increase cell migration speed on fibronectin/fibrinogen composite cables.Cell Motil Cytoskeleton,2000,46:6-16.
34.Torigoe K,Awaya A.A newly synthesized neurotropic pyrimidine compound,MS-818,may activate migratory Schwann cells in peripheral nerve regeneration.Brain Res,1998,787:337-340.
35.李强,李民,伍亚民.物理因子影响周围神经再生的研究概况.中华物理医学与康复杂志,2003,25:186-187.
36.Dubey N,Letoumeau PC,Tranquillo RT.Guided neurite elongation and Schwann cell invasion into magnetically aligned collagen in simulated peripheral nerve regeneration.Exp Neurol,1999,158:338-350.
37.Yin Q,Kemp GJ,Frostick SP.Neurotrophins,neurones and peripheral nerve regeneration.J Hand Surg,1998,23:433-437.
38.Wheeler EF,Naftel JP,Pan M,et al.Neurotrophin receptor expression is induced in a subpopulation of trigeminal neurons that label by retrograde transport of NGF or fluoro-gold following tooth injury[J].Brain Res Mol Brain Res,1998,61:23-38.
39.Frostick SP,Yin Q,Kemp GJ.Schwann cells,neurotrophic factors,and peripheral nerve regeneration[J].Microsurgery,1998,18:397-405.
40.Semkova I,Krieglstein J.Neuroprotection mediated via neurotrophic factors and induction of neurotrophic factors[J].Brain Res Brain Res Rev,1999,30:176-188.
41.Salka T,Senhe E,Noguchi I,et al.Effects of nerve crush and transection on mRNA levels for growth factor in the facial motoneurons[J].Molec Brain Res,1991,9:157.
42.Newman JP,Verity AN,Hawatmeh S,et al.Ciliary neurotrophic factor enhanace peripheral nerve regeneration.Arch Otolaryngol Head Neck Surg,1996,122:399403.
43.Inoue T,Hosokawa M,Morigiwa K,et al.Bcl-2 overexpression does not enhance in vivo axonal regeneration of retinal ganglion cells after peripheral nerve transplantation in adult mice[J].J Neurosci,2002,22:4468-4477.
44.Hammarberg H.Piehl F.Cullheim S,et al.GDNF mRNA in Schwann cells and DRG satellite ceil after chronic sciatic nerve injury.Neuroreport,1996,7:857-860.
45.Ronatd WO,Lucien J,James E,et al.Developing motor neurons rescued from programmed and axotomy-induced cell death by glial cell line-derived neurotrophic factor.Nature,1995,373:344.
46.Grothe C,Meisinger C,Herrenstein A,et aL.Expression of fibroblast growth factor-2 and fibroblast growth factor-1 messenger RNAs in Spinal ganglia and sciatic nerve:regeneration after peripheral nerve lesion.Neurosci,1996,76:123-35.
47.王非,翁龙江,李主一.碱性成纤维细胞生长因子促进神经再生的研究进展.中华显微外科杂志,1999,22:76.
48.Dezawa M,Adachi-Usami E.Role of Schwann cells in retinal ganglion cell axon regeneration[J].Prog Retin Eye Res,2000,19:171-204.
49.Lohof AM,Lp nY,Poo MM,et al.Potentiation of developing neuromuscular synapses by neurotrophins NT-3 and BDNF[J].Nature,1993,363:350-353.
50.David BP,Ambily B.Krox-20 inhibits Jun-NH2-terminal kinase/c-Jun to control Schwann cell proliferation and death[J].J CellBiol,2004,164:385-394.
51.朱浩,丁文龙.施旺细胞发育及其调控的信号通路[J].解剖科学进展,2006,12:376-380.
52.Frohnert PW,StonecypherMS,Carroll SL.Constitutive activation of the neuregulin-1 /erbB receptor signaling pathway is essential for the proliferation of a neoplastic Schwann cell line[J].Glia,2003,43:104-118.
53.Fallon KB,Havlioglu N,Hamilton LH,et al.Constitutive activation of the neuregulin-1/erbB signaling pathway promotes the proliferation of a human peripheral neuroepithelioma cell line[J].J Neurooncol,2004,66:273-284.
54.张继东,候铁胜,赵定麟.太剂量甲基强的松龙对大鼠损伤脊髓中神经生长因子的影响[J].中华外科杂志,1995,12:727.
55.Nachemson AK,Lundborg G,Myrhage R,et al.Nerve regeneration and pharmacological suppression of the scar reaction at the suture site.An experimental study on the effect of estrogen-progesterone,methylprednisoloneacetate and cis-hydroxyproline in rat sciatic nerve.Scand J Plast Reconstr Surg,1985,19:255-260.
56.姜保国,党育,张殿英等.甲基泼尼松龙对周围神经修复的影响[J].中华外科 杂志,2001,39:476-479.
57.党育,姜保国,张殿英等.甲基泼尼松龙局部应用对周围神经修复的影响[J].中华实验外科杂志,2002,19(增刊):139-140.
58.党育,姜保国,张殿英等.甲基泼尼松龙局部应用对周围神经损伤修复后的形态学研究[J].中华手外科杂志,2005,10:314-316.
59.蔡鸿敏,吴学建.甲基泼尼松龙对体外培养兔雪旺细胞迁移及增殖的影响[J].郑州大学学报(医学版),2008,43:312-314.
60.万虹,孙梅珍.雪旺氏细胞培养方法的比较[J].中国危重病急救医学2001,13(9):530-532.
61.翁惠兰,孙淑明等.雪旺细胞体外培养和纯化的新方法[J].中国基层医药,2005,12:1299-1302.
62.Friedenstein AJ.Precursor cells of mesenchymal[J].Int Rev Cytol 1976,47:327-55.
63.张培训,姜保国.骨髓间充质干细胞的研究进展与周围神经修复[J].中国临床康复杂志,2004,8:326-7.
64.吴志伟,陈峥嵘等.人胚雪旺细胞组织工程神经修复坐骨神经缺损的实验研究[J].中国临床医学,2006,13:810-3.