ROCKII特异抑制性小分子多肽促进新生大鼠DRGN_s轴突再生的体外实验研究
|
摘要
目的：(1)观察脂质体介导Rho-ROCKII特异抑制性小分子多肽转染大鼠背根节神经元(DRGNs)可行性及其转染效率；(2)观察Rho-ROCKⅡ特异性抑制的小分子多肽对大鼠背根节神经轴突再生和延长的影响。(3)对比Rho-ROCKⅡ特异性抑制剂Y27632与Rho-ROCKⅡ特异抑制性小分子多肽对大鼠背根节神经轴突再生和延长的影响。方法：(1)随机选取符合条件的健康雌性SD成年大鼠10只,按WD法制成SD大鼠T9平面完全性截瘫的脊髓损伤动物模型,完成造模7d后取T8-10节段脊髓制备脊髓提取液。(2)新生SD大鼠(＜5d)DRGNs分离、培养、鉴定、纯化、扩增。(3)观察最高转染率条件下,不同脂质体/多肽浓度对DRGNs神经轴突长度、TubulinβⅢ阳性表达以及轴突远端平均荧光密度的影响。(4)瘫痪脊髓提取液+DRGNs分别与脂质体/多肽和Y26732共同培养,观察对DRGNs神经轴突长度、TubulinβⅢ阳性表达以及轴突远端平均荧光密度的影响。结果：(1)转染后2天,不同脂质体/多肽浓度下,每组测量60个轴突长度均数为：A组(1ug多肽+2.5μl脂质体)为197±14um,B组(2ug多肽+5μl脂质体)为301±17um,C组(4ug多肽+10μl脂质体)为316±25um,D组(8ug多肽+20μl脂质体)为367±29um,E组(10ug多肽+20 u 1脂质体)为265±16um,F组(12ug多肽+30 u l脂质体)为291±21um。差异有统计学意义,D组最长,两两分析D组与其它组均有统计学差异(P<0.05)；各组TubulinβⅢ免疫荧光强度均值：A-F组分别为197.8±20.1,231.8±19.1,249.8±22.8,289.1±17.1,269.8±21.6,271.6±16.2AFU/um；其中D组(使用8ug多肽+20μl脂质体)轴突远端平均荧光密度高于其它各组,但各组间无统计学意义(P>0.05),各组神经轴突干均有细长生长锥形成,神经轴突延伸,神经轴突干TubulinβⅢ浓聚,生长锥增粗。(2)瘫痪脊髓提取液+DRGNs分别与脂质体/多肽和Y26732共同培养,观察各组测量60个轴突长度均数：A组(DRGNs+PBS)为391±20um,B组(DRGNs+完全性截瘫大鼠脊髓提取液)为107±21um,C组(DRGNs+完全性截瘫大鼠脊髓提取液+脂质体)为121±23um,D组(DRGNs+完全性截瘫大鼠脊髓提取液+Y26732)为307±16um, E组(DRGNs+完全性截瘫大鼠脊髓提取液+脂质体+抑制性小分子多肽)为367±19um,经方差分析,各组差异具有显著性统计学意义(P<0.0001),两两比较,B、C组与A、D、E组之间P<0.05,均有明显统计学意义；B、C两组之间P>0.05,无统计学意义。D、E组之间P<0.05,有统计学意义。轴突远端平均荧光密度分别是：A组为183.4±3.7 AFU/um,B组为56.1±4.2AFU/um,C组为62.7±5.9AFU/um,D组为278.1±6.2AFU/um,E组为313.4±3.6 AFU/um。经方差分析,各组差异具有显著性统计学意义(P<0.0001),两两比较,A、D、E组与B、C组差别均有显著统计学意义(P<0.0001),而B、C组之间无明显统计学差异(P>0.05),E组与A、D组差别有统计学意义(P<0.05)。其荧光密度高,TubulinβⅢ阳性表达强与其他各组。结论：(1)脂质体介导的Rho-ROCKⅡ特异抑制性多肽转染大鼠背根节神经(DRGNs)可促进轴突生长,形成多个轴突或明显轴突分支,使用8ug抑制性多肽20μl脂质体浓度时轴突再生作用更明显。(2)适当浓度的Rho-ROCKⅡ特异抑制性多肽/脂质体转染新生大鼠背根节神经元DRGNs和Y27632均可促进轴突延长,脂质体介导的抑制性多肽转染DRGNs比Y27632干预作用更明显。
Objective:(1) To explore the transfection efficiency of that the inhibitory polypeptide of Rho-ROCKII was introduced into dorsal root ganglia neurons(DRGNs) in vitro with Lipofectamine 2000 and the optimized factors affecting the transfection efficiency of liposome. (2) To observe the effect on DRGNs axonal growth and extension with application of Inhibitory polypeptide of Rho-ROCKII in vitro. (3) To Compare the effect on DRGNs axonal growth and extension of rats treated with Y27632 and the micromolecule polypeptide. Methods:(1) 10 adult female Sprague-Dawley (SD) rats were randomly selected into a groups and were subjected to weight-drop impact causing complete paraplegia, The T8-10 spinal cord extracts (SCE) were harvested in complete paraplegia group at 7th day after spinal cord injury in rats. (2) All thoracic-lumbar DRGNs of neogenic SD rats (<5d) were harvested, and then DRNGs were harvested, indentified, Cultured, purified and amplified. (3) Based on the most optimized transfection condition (the ratio of liposome and polypeptide is 2.5:1), The axonal length, the mean fluorescence density of positive expression of TubulinβⅢat axonal distal end of DRGNs were observed at different concerations of liposome/inhibitory polypeptide respectively. (4)The axonal length, the mean fluorescence density of positive expression of TubulinβⅢat axonal distal end of DRGNs were observed in vitro after co-culture with complete paralysis SCE+ Y26732 or liposome/ inhibitory polypeptide at 2 days respectively. Result:(1)The mean lengths of neuronal axons of neogenesis DRGNs was 197±14um in group A, 301±17um in group B,316±25um in group C,367±29um in group D,265±16um in group E and291±21um in group F respectively at 2 days after co-culture. The axonal length in group D was significantly longer compared to group A, B, C and E respectively.there was no difference among group A,B, C and E. The mean fluorescence densities at axonal distal end were 197.8±20.1 AFU/um in group A,231.8±19.1AFU/um in group B,249.8±22.8 AFU/um in group C,289.1±17.1 AFU/um in group D,269.8±21.6AFU/um in group E and 271.6.8±16.2 AFU/um in group F respecviely at 2 days after co-culture.The mean fluorescence densities in group D was significantly stronger than it in group A,B, C and E and there is no significant difference among group A,B, C and E. (2) The mean lengths of neuronal axons of neogenesis DRGNs was391±20um in group A (DRGNs+PBS),107±21 um in group B (DRGNs+ complete paralysis SCE),121±23um in group C (DRGNs+complete paralysis SCE+liposome),307±16um in group D (DRGNs+complete paralysis SCE+Y26732) and 367±19um in group E (DRGNs+complete paralysis SCE+liposome+inhibitory polypeptide)respectively at 2 days after co-culture. The axonal length in group B and group C were shorter than it in group A,D,and E, and group E was longer than group D, there was no difference between the groups B and C. The mean fluorescence densities at axonal distal end were 183.4±3.7AFU/um in group A,56.1±4.2AFU/um in group B, 62.7±5.9 AFU/um in group C,278.1±6.2AFU/um in group D and 313.4±3.6AFU/um in group E in group F respecviely at 2 days after co-culture. The mean fluorescence densities at axonal distal end in groupA,D,E were stronger than groupB, C and group E was stronger than group A,D, there was no difference between the groups B and C. Conclusion:(1) The inhibitory polypeptide of Rho-ROCKⅡtransfectd by Lipofectamine 2000 can promote axon extend, induce regeneration of axon branches. The highest efficiency was achieved in the condition of 8ug polypeptide and 20μl liposome. (2) The effect of promoting axon extend of the inhibitory polypeptide is significantly stronger than Y27632.
Objective:(1) To explore the transfection efficiency of that the inhibitory polypeptide of Rho-ROCKII was introduced into dorsal root ganglia neurons(DRGNs) in vitro with Lipofectamine 2000 and the optimized factors affecting the transfection efficiency of liposome. (2) To observe the effect on DRGNs axonal growth and extension with application of Inhibitory polypeptide of Rho-ROCKII in vitro. (3) To Compare the effect on DRGNs axonal growth and extension of rats treated with Y27632 and the micromolecule polypeptide. Methods:(1) 10 adult female Sprague-Dawley (SD) rats were randomly selected into a groups and were subjected to weight-drop impact causing complete paraplegia, The T8-10 spinal cord extracts (SCE) were harvested in complete paraplegia group at 7th day after spinal cord injury in rats. (2) All thoracic-lumbar DRGNs of neogenic SD rats (<5d) were harvested, and then DRNGs were harvested, indentified, Cultured, purified and amplified. (3) Based on the most optimized transfection condition (the ratio of liposome and polypeptide is 2.5:1), The axonal length, the mean fluorescence density of positive expression of TubulinβⅢat axonal distal end of DRGNs were observed at different concerations of liposome/inhibitory polypeptide respectively. (4)The axonal length, the mean fluorescence density of positive expression of TubulinβⅢat axonal distal end of DRGNs were observed in vitro after co-culture with complete paralysis SCE+ Y26732 or liposome/ inhibitory polypeptide at 2 days respectively. Result:(1)The mean lengths of neuronal axons of neogenesis DRGNs was 197±14um in group A, 301±17um in group B,316±25um in group C,367±29um in group D,265±16um in group E and291±21um in group F respectively at 2 days after co-culture. The axonal length in group D was significantly longer compared to group A, B, C and E respectively.there was no difference among group A,B, C and E. The mean fluorescence densities at axonal distal end were 197.8±20.1 AFU/um in group A,231.8±19.1AFU/um in group B,249.8±22.8 AFU/um in group C,289.1±17.1 AFU/um in group D,269.8±21.6AFU/um in group E and 271.6.8±16.2 AFU/um in group F respecviely at 2 days after co-culture.The mean fluorescence densities in group D was significantly stronger than it in group A,B, C and E and there is no significant difference among group A,B, C and E. (2) The mean lengths of neuronal axons of neogenesis DRGNs was391±20um in group A (DRGNs+PBS),107±21 um in group B (DRGNs+ complete paralysis SCE),121±23um in group C (DRGNs+complete paralysis SCE+liposome),307±16um in group D (DRGNs+complete paralysis SCE+Y26732) and 367±19um in group E (DRGNs+complete paralysis SCE+liposome+inhibitory polypeptide)respectively at 2 days after co-culture. The axonal length in group B and group C were shorter than it in group A,D,and E, and group E was longer than group D, there was no difference between the groups B and C. The mean fluorescence densities at axonal distal end were 183.4±3.7AFU/um in group A,56.1±4.2AFU/um in group B, 62.7±5.9 AFU/um in group C,278.1±6.2AFU/um in group D and 313.4±3.6AFU/um in group E in group F respecviely at 2 days after co-culture. The mean fluorescence densities at axonal distal end in groupA,D,E were stronger than groupB, C and group E was stronger than group A,D, there was no difference between the groups B and C. Conclusion:(1) The inhibitory polypeptide of Rho-ROCKⅡtransfectd by Lipofectamine 2000 can promote axon extend, induce regeneration of axon branches. The highest efficiency was achieved in the condition of 8ug polypeptide and 20μl liposome. (2) The effect of promoting axon extend of the inhibitory polypeptide is significantly stronger than Y27632.
引文
1. Schwab ME, Nogo and axon regeneration[J].Curr Opin Neurobiol,2004, 14(1):118-124.
2. Hurbert R J. Strategies of medical intervention in the management of acute spinal cord injury [J]. Spine,2006,31(11):16-21
3. Rossignol S, Schwab M, Schwartz M, et al. Spinal cord injury:time to move. The Journal of Neuroscience,2007,27(44):11782-11792
4. Cafferty WB, McGee AW, Strittmatter SM. Axonal growth therapeutics: regeneration or sprouting or plasticity.Trends Neurosci,2008,31(5): 215-220
5. Nishio T.Axonal regeneration and neural network reconstruction in mammalian CNS. J Neurol,2009,256 (Suppl 3):306-309.
6. Fenrich K, Gordon T. Canadian Association of Neuroscience review: axonal regeneration in the peripheral and central nervous systems:current issues and advances. Can J Neurol Sci,2004,31:142-156.
7. Yiu G, He Z. Glial inhibition of CNS axon regeneration[J].Nat Rev Neurosci,2006,7 (8):617-627.
8. Hata K, Fujitani M, Yasuda Y,et al.RGMa inhibition promotes axonal growth and recovery after spinal cord injury. The Journal of Cell Biology, 2006,173(1):47-58
9. Liu X, Hashimoto M, Horii H, et al.Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury. Biochem Biophys Res Commun,2009,15;382(4):795-800
10. Sayas C.L,Ariaens A,Ponsioen B,et al.GSK-3 is activiated by the tyrosine kinase Pyk2 during LPA1-mediated neurite restraction.Molecular Biology of the Cell,2006,17(4):1834-1844
11. Gopalakrishnan SM, Teusch N, Imhof C, et al. Role of Rho kinase pathway in chondroitin sulfate proteoglycan—mediated inhibition of neurite outgrowth in PC 12 cells.J Neurosci Res,2008,86(10):2214-2226
12. Buss A, Pech K, Kakulas BA, et al. NG2 and phosphacan are present in the astroglial SCar after human traumatic spinal cord injury. BMCNeurol,2009,9(32):1-15
13. Chan CC, Robe,s CR,Steeves JD, et al. Aggrecan components differentially modulate nerve growth factor-responsive and neurotrop-hin-3-responsive dorsal root ganglion neurite growth. [J] J Neurosci Res,2008,86(3):581-592
14. Fabes J, Anderson P,Brennan C, et al. Regeneration—enhancing effects of EphA4 blocking peptide following corticospinal tract injury in adultrat spinal cord. Eur J Neurosci,2007,26(9):2496-2505
15. Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders.Nat Rev Drug Discov,2005,4(5):387-398
16. Kuto T,Yamaguchi A,Lwata T,et al.The therapeutic effects of the Rho-ROCK inhibitors on the CNS disorders.Therapeutics and Clinical Risk Management,2008:4(3):605-615
17. Gensel JC,Tovar CA,Hamers FP,et al.Behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats[J] .J Neurotrauma,2006,23(1):36-54
18. Erschbamer MK,Hofstetter CP,Olson L. RhoA, RhoB, RhoC, Rac1, Cdc42, and Tc10 mRNA Levels in Spinal Cord, Sensory Ganglia, and Corticospinal Tract Neurons and Long-Lasting Specific Changes Following Spinal Cord Injury[J].J Comp Neurol,2005,484(2):224-233
19.胡俊勇,李佛保,李先明,廖威明,傅明.大鼠损伤脊髓提取液对骨髓基质细胞培养特性的影响[J].中国脊柱脊髓杂志,2003,13(19)：530-533.
20. Basso DM,Beattie MS,Bresnahan JC.A sensitive and reliable locomotor rating scale for open field testing in rats[J]. J Neurotrauma,1995, 12(1):1-12
21. Zhou FQ, Zhou J, Dedhar S.NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC[J].Neuron,2004,42(6):897-912
22. He Y, Yu W, Baas PW. Microtubule reconfiguration during axonal retraction induced by nitric oxide [J]. J Neurosci,2002,22(14): 5982-5991
23. Conrad S, Schluesener HJ, Trautmann K, etal.Prolonged lesional expression of RhoA and RhoB following spinal cord injury. J Comp Neurol,2005,487(2):166-175.
24. Buss A, Pech K, Kakulas BA,et al.NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury. BMC Neurol, 2009,9(32):1-15
25. Chan CC, Roberts CR, Steeves JD, et al. Aggrecan components differentially modulate nerve growth factor-responsive and neurotrophin-3-responsive dorsal root ganglion neurite growth.J Neurosci Res,2008,86(3):581-592
26. Gopalakrishnan SM, Teusch N, Imhof C, et al.Role of Rho kinase pathway in chondroitin sulfate proteoglycan-mediated inhibition of neurite outgrowth in PC 12 cells. J Neurosci Res,2008,86(10):2214-2226
27. Jain A, Brady-Kalnay SM, Bellamkonda RV..Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension. J Neurosci Res,2004,15,77(2):299-307
28. Lingor P, Teusch N, Schwarz K, et al.Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. J Neurochem, 2007,103(1):181-189
29. Dickson B. Molecular Mechanisms of Axon Guidance. Science,2002, 298 (5600):1959-1963
30. Bolsover S, Fabes J, Anderson PN.Axonal guidance molecules and the failure of axonal regeneration in the adult mammalian spinal cord. Restor Neurol Neurosci,2008;26(2-3):117-130
31. Fabes J, Anderson P, Brennan C, et al.Regeneration-enhancing effects of EphA4 blocking peptide following corticospinal tract injury in adult rat spinal cord. Eur J Neurosci,2007,26(9):2496-2505
32. Benson MD, Romero MI, Lush ME, et al.Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc Natl Acad Sci USA,2005,102(30): 10694-10709
33. 黄贻泽,冯大雄,李骏等.Y27632对脊髓损伤微环境下新生大鼠背根节神经元轴突再生的影响[J].中国脊柱脊髓杂志,2010,(09)20：765-770
34. Kubo A, Hata K, Yamaguchi A, et al. Rho-ROCK inhibitors as emerging strategies to promote nerve regeneration [J].Curr Pharml Des,2007, 13(24):2493-2499
35. Park JB,Yiu G,Kaneko S.A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors [J].Neuron,2005,45(3):345-351
36. Dergham CI, Winton MJ, Mckerracher L. Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system [J]. J Cell Biol,2003,162(2):233-243
37. Rosner H, Moller W, Wassermann T,et al.Attenuation of actinomyosin II contractile activity in growth cones accelerates filopodia-guided and microtubule-based neurite elongation. Brain Res,2007,1176 (24):1-10
38. Bito H, Furuyashiki T, Ishihara H,. A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons. Neuron,2000,26(2):431-441
39. Borisoff JF, Chan CC, Hiebert GW, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates [J]. Mol Cell Neurosci,2003,22(3):405-416
40. Chan CC, Khodarahmi K, Liu J,et al.Dose-dependent beneficial and detrimental effects of ROCK inhibitor Y27632 on axonal sprouting and functional recovery after rat spinal cord injury.Exp Neurol,2005,196(2): 352-364
41. Sung JK,et al.A possible roe of RhoA/Tho-kinase in experimental spinal cord injury in rat[J].Brain Res,2003,959 (1):29-38
42. Tanalia H,et al.Cytoplasmic p21(Cip1/WAF1) enhances axonal regeneration and functional recovery after spinal cord injury in rats[J].Neurosci,2004,127(1):155-164
43. Sistiabudi Rizaldi, Ivanisevic Albena. Patterning of polypeptides on a collagen—terminated tissue surface[J]. Journal of Physics and Chemistry C,2007,111(31):11676-11681.
44. Sun Jing, Chen Xuesi, Deng Chao.et al. Direct formation of giant vesicles from synthetic polypeptides[J]. Langmuir,2007,23(16):8308—8315.
45. Cudic Mare, Mari Frank, Fields Gregg B. Synthesis and solidph-ase application of suitably protected-hydroxyvaline building blocks[J]. Journal of Organic Chemistry,2007,72(15):5581-5586.
46. Willy Agut, Daniel Taton, Sebastien Lecommandoux. A versatile synthetic approach to polypeptide based rod-coil block copolymers by click chemistry[J]. Macromolecules,2007,40(16):5653-5661.
47. Kisiday J,Jin M,Kurz B,et al.Proc.Natl.Acad.Sei.USA,2002,99:9996.
48. Trauger J W,Lin F-F,Tirner M S,et al.Kinetic mechanism for Human Rho-Kinase II(ROCK-II).Biochemistry,2002,41:8948-8953
49. Grandpre T,LI S,Strittmatter.Nogo-66 receptor antagonist peptide promote axonal regeneration.Nature,2002,417:547-550
50. Rubanyi G M,The future of human gene therapy[J].Mol Aspects Med, 2001,22(3):113-142.
51. Xu R,Harrison P M,Chen M,et al.Cytoglobin overexpression protects against damage-induced fibrosis[J]. Mol Ther,2006,13(6):1093-1100.
52. Yosuke Obata,,aisuke S,Shinji T.Bioeonjugate Chem[J],2008,19(5) 1055-1063.
53.姜云霞,王冰,崔韶晖,等.安徽农业科学[J],2009,(17)：7862-7864.
54. Almofti MR,Harashima H,Shinohara Y,,et al.Arch Biochem Biophys [J], 2003,410(2):246-253.
55.陈梦飞,陆大祥,戚仁斌,等.中国病理生理杂志[J],2008,24(7)：1254-1258.
56.张宏波,项琪,赵文等.多肽、蛋白类药物脂质体研究进展[J]中国生物工程杂志,2007,27,(06)：101-106
57. Takeshi sunallli, Kanetomo sato, Tomoaki Matsuura, et al. Femtoliter compartment in liposomes for in vitro selection of proteins. Analytical Biochemistry.2006,357:128-136
58. Ma B, Zhang S, Jiang H.et al.Lipoplex morphologies and their influences on tranfection efficiency in gene delivery[J].JControl Release, 2007.123(3):184-194.
59.陈彦样.乔卫红,刘栋良,等.阳离子脂质体的转染机制及转染效率影响因素[J].生物工程学报,2007,23(5)：776-780.
60. Wzasungu L, Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes[J]. J Control Release,2006,116(2):255-264.
61. Nguyen LT, Atobe K, BarichelloJM, et al. Complex formation with plasmid DNA increases the cytotoxicity of cationic lipoooraes [J]. Biol Hmrm Bull,2007,30(4):751-757.
62. Dass CR. Lipoplex-mediated delivery of nucleic acids:factors affecting in vivo transfection [J]. J Mol Med,2004,82(9); 579-591
63. Dalby B.Cates S,Harris A,et al.Advanced transfection with Lipofectamine 2000 reagent:primary neurons,siRNA,and high-throughput applications [J]. Methods,2004,33(2):95-103.
64. Eastman SJ, Siegel C, Tousignant J, et al. Biophysical characterization of cationic lipid:DNA complexes [J]. Bioehim Biophys Acta,1997, 1325(1):41-62.
65. Dodds E, Dunckley MG. Naujoks K, et al. lipofection of cultured mouse muscle cells:a direct comparison of Lipofectamine and DOSPER [J]. Gene Ther,1998,5(4):542-551.
66. Nguyen LT, Atobe K, BarichelloJM, et al. Complex formation with plasmid DNA increases the cytotoxicity of cationic lipoooraes[J]. Biol Hmrm Bull,2007,30(4):751-757.
1. Rossignol S, Schwab M, Schwartz M, et al. Spinal cord injury:time to move? The Journal of Neuroscience,2007,27(44):11782-11792
2. Cafferty WB, McGee AW, Strittmatter SM. Axonal growth therapeutics: regeneration or sprouting or plasticity? Trends Neurosci,2008,31(5): 215-20
3. Nishio T.Axonal regeneration and neural network reconstruction in mammalian CNS. J Neurol,2009,256 (Suppl 3):306-309.
4. Conrad S, Schluesener HJ, Trautmann K, etal.Prolonged lesional expression of RhoA and RhoB following spinal cord injury. J Comp Neurol,2005,487 (2):166-75.
5. Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders.Nat Rev Drug Discov,2005,4(5):387-398
6. Kuto T,Yamaguchi A,Lwata T,et al.The therapeutic effects of the Rho-ROCK inhibitors on the CNS disorders.Therapeutics and Clinical Risk Management,2008:4(3):605-615
7. Hata K, Fujitani M, Yasuda Y,et al.RGMa inhibition promotes axonal growth and recovery after spinal cord injury. The Journal of Cell Biology, 2006,173(1):47-58
8. Liu X, Hashimoto M, Horii H, et al.Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury. Biochem Biophys Res Commun,2009,15;382(4):795-800
9. Kwok JC, Afshari F, Garcia-Alias G,et al.Proteoglycans in the central nervous system:plasticity, regeneration and their stimulation with chondroitinase ABC. Restor Neurol Neurosci,2008,26(2-3):131-145
10. Buss A, Pech K, Kakulas BA,et al.NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury. BMC Neurol, 2009,9(32):1-15
11. Chan CC, Roberts CR, Steeves JD,et al.Aggrecan components differentially modulate nerve growth factor-responsive and neurotrophin-3-responsive dorsal root ganglion neurite growth. J Neurosci Res,2008,86(3):581-592
12. Gopalakrishnan SM, Teusch N, Imhof C, et al.Role of Rho kinase pathway in chondroitin sulfate proteoglycan-mediated inhibition of neurite outgrowth in PC12 cells. J Neurosci Res,2008,86(10):2214-2226
13. Lingor P, Teusch N, Schwarz K, et al.Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. J Neurochem, 2007,103(1):181-189
14. Feng-quan Zhou, walzer M, Yao-hong Wu, et al. Neurophins support regenerative axon assembly over CSPGs by an ECM-integrin-independent mechanism. Journal of Cell Science,2006,119 (13):2787-2796
15. Dickson B. Molecular Mechanisms of Axon Guidance. Science,2002,298 (5600):1959-1963
16. Bolsover S, Fabes J, Anderson PN.Axonal guidance molecules and the failure of axonal regeneration in the adult mammalian spinal cord. Restor Neurol Neurosci,2008;26(2-3):117-130
17. Gallo G. RhoA-kinase coordinates F-actin organization and myosin Ⅱ activity during semaphorin-3A-induced axon retraction. J Cell Sci, 2006,119(Pt 16):3413-3423
18. Fabes J, Anderson P, Brennan C, et al.Regeneration-enhancing effects of EphA4 blocking peptide following corticospinal tract injury in adult rat spinal cord. Eur J Neurosci,2007,26(9):2496-2505
19. Benson MD, Romero MI, Lush ME, et al.Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc Natl Acad Sci USA,2005,102(30): 10694-10709
20. Sayas C.L,Ariaens A,Ponsioen B,et al.GSK-3 is activiated by the tyrosine kinase Pyk2 during LPA1-mediated neurite restraction.Molecular Biology of the Cell,2006,17(4):1834-1844
21. Conrad S, Schluesener HJ, Trautmann K, etal.Prolonged lesional expression of RhoA and RhoB following spinal cord injury. J Comp Neurol,2005 487(2):166-75.
22. Duffy P, Schmandke A, Schmandke A,et al.Rho-associated kinase Ⅱ (ROCKⅡ) limits axonal growth after trauma within the adult mouse spinal cord. J Neurosci,2009,29(48):15266-15276
23. Wood-Kaczmar A, Kraus M, Ishiguro K,et al.An alternatively spliced form of glycogen synthase kinase-3β is targeted to growing neurites and growth cones. Mol Cell Neurosci,2009,42(3):184-194
24. Dill J, Wang H, Zhou F,et al.Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS. J Neurosci.2008,28(36): 8914-892
25. Trivedi N, Marsh P, Goold RG,et al.Glycogen synthase kinase-3p phosphorylation of MAP IB at Ser1260 and Thr1265 is spatially restricted to growing axons.J Cell Sci,2005,118(Pt 5):993-1005
2. Hurbert R J. Strategies of medical intervention in the management of acute spinal cord injury [J]. Spine,2006,31(11):16-21
3. Rossignol S, Schwab M, Schwartz M, et al. Spinal cord injury:time to move. The Journal of Neuroscience,2007,27(44):11782-11792
4. Cafferty WB, McGee AW, Strittmatter SM. Axonal growth therapeutics: regeneration or sprouting or plasticity.Trends Neurosci,2008,31(5): 215-220
5. Nishio T.Axonal regeneration and neural network reconstruction in mammalian CNS. J Neurol,2009,256 (Suppl 3):306-309.
6. Fenrich K, Gordon T. Canadian Association of Neuroscience review: axonal regeneration in the peripheral and central nervous systems:current issues and advances. Can J Neurol Sci,2004,31:142-156.
7. Yiu G, He Z. Glial inhibition of CNS axon regeneration[J].Nat Rev Neurosci,2006,7 (8):617-627.
8. Hata K, Fujitani M, Yasuda Y,et al.RGMa inhibition promotes axonal growth and recovery after spinal cord injury. The Journal of Cell Biology, 2006,173(1):47-58
9. Liu X, Hashimoto M, Horii H, et al.Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury. Biochem Biophys Res Commun,2009,15;382(4):795-800
10. Sayas C.L,Ariaens A,Ponsioen B,et al.GSK-3 is activiated by the tyrosine kinase Pyk2 during LPA1-mediated neurite restraction.Molecular Biology of the Cell,2006,17(4):1834-1844
11. Gopalakrishnan SM, Teusch N, Imhof C, et al. Role of Rho kinase pathway in chondroitin sulfate proteoglycan—mediated inhibition of neurite outgrowth in PC 12 cells.J Neurosci Res,2008,86(10):2214-2226
12. Buss A, Pech K, Kakulas BA, et al. NG2 and phosphacan are present in the astroglial SCar after human traumatic spinal cord injury. BMCNeurol,2009,9(32):1-15
13. Chan CC, Robe,s CR,Steeves JD, et al. Aggrecan components differentially modulate nerve growth factor-responsive and neurotrop-hin-3-responsive dorsal root ganglion neurite growth. [J] J Neurosci Res,2008,86(3):581-592
14. Fabes J, Anderson P,Brennan C, et al. Regeneration—enhancing effects of EphA4 blocking peptide following corticospinal tract injury in adultrat spinal cord. Eur J Neurosci,2007,26(9):2496-2505
15. Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders.Nat Rev Drug Discov,2005,4(5):387-398
16. Kuto T,Yamaguchi A,Lwata T,et al.The therapeutic effects of the Rho-ROCK inhibitors on the CNS disorders.Therapeutics and Clinical Risk Management,2008:4(3):605-615
17. Gensel JC,Tovar CA,Hamers FP,et al.Behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats[J] .J Neurotrauma,2006,23(1):36-54
18. Erschbamer MK,Hofstetter CP,Olson L. RhoA, RhoB, RhoC, Rac1, Cdc42, and Tc10 mRNA Levels in Spinal Cord, Sensory Ganglia, and Corticospinal Tract Neurons and Long-Lasting Specific Changes Following Spinal Cord Injury[J].J Comp Neurol,2005,484(2):224-233
19.胡俊勇,李佛保,李先明,廖威明,傅明.大鼠损伤脊髓提取液对骨髓基质细胞培养特性的影响[J].中国脊柱脊髓杂志,2003,13(19)：530-533.
20. Basso DM,Beattie MS,Bresnahan JC.A sensitive and reliable locomotor rating scale for open field testing in rats[J]. J Neurotrauma,1995, 12(1):1-12
21. Zhou FQ, Zhou J, Dedhar S.NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC[J].Neuron,2004,42(6):897-912
22. He Y, Yu W, Baas PW. Microtubule reconfiguration during axonal retraction induced by nitric oxide [J]. J Neurosci,2002,22(14): 5982-5991
23. Conrad S, Schluesener HJ, Trautmann K, etal.Prolonged lesional expression of RhoA and RhoB following spinal cord injury. J Comp Neurol,2005,487(2):166-175.
24. Buss A, Pech K, Kakulas BA,et al.NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury. BMC Neurol, 2009,9(32):1-15
25. Chan CC, Roberts CR, Steeves JD, et al. Aggrecan components differentially modulate nerve growth factor-responsive and neurotrophin-3-responsive dorsal root ganglion neurite growth.J Neurosci Res,2008,86(3):581-592
26. Gopalakrishnan SM, Teusch N, Imhof C, et al.Role of Rho kinase pathway in chondroitin sulfate proteoglycan-mediated inhibition of neurite outgrowth in PC 12 cells. J Neurosci Res,2008,86(10):2214-2226
27. Jain A, Brady-Kalnay SM, Bellamkonda RV..Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension. J Neurosci Res,2004,15,77(2):299-307
28. Lingor P, Teusch N, Schwarz K, et al.Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. J Neurochem, 2007,103(1):181-189
29. Dickson B. Molecular Mechanisms of Axon Guidance. Science,2002, 298 (5600):1959-1963
30. Bolsover S, Fabes J, Anderson PN.Axonal guidance molecules and the failure of axonal regeneration in the adult mammalian spinal cord. Restor Neurol Neurosci,2008;26(2-3):117-130
31. Fabes J, Anderson P, Brennan C, et al.Regeneration-enhancing effects of EphA4 blocking peptide following corticospinal tract injury in adult rat spinal cord. Eur J Neurosci,2007,26(9):2496-2505
32. Benson MD, Romero MI, Lush ME, et al.Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc Natl Acad Sci USA,2005,102(30): 10694-10709
33. 黄贻泽,冯大雄,李骏等.Y27632对脊髓损伤微环境下新生大鼠背根节神经元轴突再生的影响[J].中国脊柱脊髓杂志,2010,(09)20：765-770
34. Kubo A, Hata K, Yamaguchi A, et al. Rho-ROCK inhibitors as emerging strategies to promote nerve regeneration [J].Curr Pharml Des,2007, 13(24):2493-2499
35. Park JB,Yiu G,Kaneko S.A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors [J].Neuron,2005,45(3):345-351
36. Dergham CI, Winton MJ, Mckerracher L. Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system [J]. J Cell Biol,2003,162(2):233-243
37. Rosner H, Moller W, Wassermann T,et al.Attenuation of actinomyosin II contractile activity in growth cones accelerates filopodia-guided and microtubule-based neurite elongation. Brain Res,2007,1176 (24):1-10
38. Bito H, Furuyashiki T, Ishihara H,. A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons. Neuron,2000,26(2):431-441
39. Borisoff JF, Chan CC, Hiebert GW, et al. Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates [J]. Mol Cell Neurosci,2003,22(3):405-416
40. Chan CC, Khodarahmi K, Liu J,et al.Dose-dependent beneficial and detrimental effects of ROCK inhibitor Y27632 on axonal sprouting and functional recovery after rat spinal cord injury.Exp Neurol,2005,196(2): 352-364
41. Sung JK,et al.A possible roe of RhoA/Tho-kinase in experimental spinal cord injury in rat[J].Brain Res,2003,959 (1):29-38
42. Tanalia H,et al.Cytoplasmic p21(Cip1/WAF1) enhances axonal regeneration and functional recovery after spinal cord injury in rats[J].Neurosci,2004,127(1):155-164
43. Sistiabudi Rizaldi, Ivanisevic Albena. Patterning of polypeptides on a collagen—terminated tissue surface[J]. Journal of Physics and Chemistry C,2007,111(31):11676-11681.
44. Sun Jing, Chen Xuesi, Deng Chao.et al. Direct formation of giant vesicles from synthetic polypeptides[J]. Langmuir,2007,23(16):8308—8315.
45. Cudic Mare, Mari Frank, Fields Gregg B. Synthesis and solidph-ase application of suitably protected-hydroxyvaline building blocks[J]. Journal of Organic Chemistry,2007,72(15):5581-5586.
46. Willy Agut, Daniel Taton, Sebastien Lecommandoux. A versatile synthetic approach to polypeptide based rod-coil block copolymers by click chemistry[J]. Macromolecules,2007,40(16):5653-5661.
47. Kisiday J,Jin M,Kurz B,et al.Proc.Natl.Acad.Sei.USA,2002,99:9996.
48. Trauger J W,Lin F-F,Tirner M S,et al.Kinetic mechanism for Human Rho-Kinase II(ROCK-II).Biochemistry,2002,41:8948-8953
49. Grandpre T,LI S,Strittmatter.Nogo-66 receptor antagonist peptide promote axonal regeneration.Nature,2002,417:547-550
50. Rubanyi G M,The future of human gene therapy[J].Mol Aspects Med, 2001,22(3):113-142.
51. Xu R,Harrison P M,Chen M,et al.Cytoglobin overexpression protects against damage-induced fibrosis[J]. Mol Ther,2006,13(6):1093-1100.
52. Yosuke Obata,,aisuke S,Shinji T.Bioeonjugate Chem[J],2008,19(5) 1055-1063.
53.姜云霞,王冰,崔韶晖,等.安徽农业科学[J],2009,(17)：7862-7864.
54. Almofti MR,Harashima H,Shinohara Y,,et al.Arch Biochem Biophys [J], 2003,410(2):246-253.
55.陈梦飞,陆大祥,戚仁斌,等.中国病理生理杂志[J],2008,24(7)：1254-1258.
56.张宏波,项琪,赵文等.多肽、蛋白类药物脂质体研究进展[J]中国生物工程杂志,2007,27,(06)：101-106
57. Takeshi sunallli, Kanetomo sato, Tomoaki Matsuura, et al. Femtoliter compartment in liposomes for in vitro selection of proteins. Analytical Biochemistry.2006,357:128-136
58. Ma B, Zhang S, Jiang H.et al.Lipoplex morphologies and their influences on tranfection efficiency in gene delivery[J].JControl Release, 2007.123(3):184-194.
59.陈彦样.乔卫红,刘栋良,等.阳离子脂质体的转染机制及转染效率影响因素[J].生物工程学报,2007,23(5)：776-780.
60. Wzasungu L, Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes[J]. J Control Release,2006,116(2):255-264.
61. Nguyen LT, Atobe K, BarichelloJM, et al. Complex formation with plasmid DNA increases the cytotoxicity of cationic lipoooraes [J]. Biol Hmrm Bull,2007,30(4):751-757.
62. Dass CR. Lipoplex-mediated delivery of nucleic acids:factors affecting in vivo transfection [J]. J Mol Med,2004,82(9); 579-591
63. Dalby B.Cates S,Harris A,et al.Advanced transfection with Lipofectamine 2000 reagent:primary neurons,siRNA,and high-throughput applications [J]. Methods,2004,33(2):95-103.
64. Eastman SJ, Siegel C, Tousignant J, et al. Biophysical characterization of cationic lipid:DNA complexes [J]. Bioehim Biophys Acta,1997, 1325(1):41-62.
65. Dodds E, Dunckley MG. Naujoks K, et al. lipofection of cultured mouse muscle cells:a direct comparison of Lipofectamine and DOSPER [J]. Gene Ther,1998,5(4):542-551.
66. Nguyen LT, Atobe K, BarichelloJM, et al. Complex formation with plasmid DNA increases the cytotoxicity of cationic lipoooraes[J]. Biol Hmrm Bull,2007,30(4):751-757.
1. Rossignol S, Schwab M, Schwartz M, et al. Spinal cord injury:time to move? The Journal of Neuroscience,2007,27(44):11782-11792
2. Cafferty WB, McGee AW, Strittmatter SM. Axonal growth therapeutics: regeneration or sprouting or plasticity? Trends Neurosci,2008,31(5): 215-20
3. Nishio T.Axonal regeneration and neural network reconstruction in mammalian CNS. J Neurol,2009,256 (Suppl 3):306-309.
4. Conrad S, Schluesener HJ, Trautmann K, etal.Prolonged lesional expression of RhoA and RhoB following spinal cord injury. J Comp Neurol,2005,487 (2):166-75.
5. Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders.Nat Rev Drug Discov,2005,4(5):387-398
6. Kuto T,Yamaguchi A,Lwata T,et al.The therapeutic effects of the Rho-ROCK inhibitors on the CNS disorders.Therapeutics and Clinical Risk Management,2008:4(3):605-615
7. Hata K, Fujitani M, Yasuda Y,et al.RGMa inhibition promotes axonal growth and recovery after spinal cord injury. The Journal of Cell Biology, 2006,173(1):47-58
8. Liu X, Hashimoto M, Horii H, et al.Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury. Biochem Biophys Res Commun,2009,15;382(4):795-800
9. Kwok JC, Afshari F, Garcia-Alias G,et al.Proteoglycans in the central nervous system:plasticity, regeneration and their stimulation with chondroitinase ABC. Restor Neurol Neurosci,2008,26(2-3):131-145
10. Buss A, Pech K, Kakulas BA,et al.NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury. BMC Neurol, 2009,9(32):1-15
11. Chan CC, Roberts CR, Steeves JD,et al.Aggrecan components differentially modulate nerve growth factor-responsive and neurotrophin-3-responsive dorsal root ganglion neurite growth. J Neurosci Res,2008,86(3):581-592
12. Gopalakrishnan SM, Teusch N, Imhof C, et al.Role of Rho kinase pathway in chondroitin sulfate proteoglycan-mediated inhibition of neurite outgrowth in PC12 cells. J Neurosci Res,2008,86(10):2214-2226
13. Lingor P, Teusch N, Schwarz K, et al.Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. J Neurochem, 2007,103(1):181-189
14. Feng-quan Zhou, walzer M, Yao-hong Wu, et al. Neurophins support regenerative axon assembly over CSPGs by an ECM-integrin-independent mechanism. Journal of Cell Science,2006,119 (13):2787-2796
15. Dickson B. Molecular Mechanisms of Axon Guidance. Science,2002,298 (5600):1959-1963
16. Bolsover S, Fabes J, Anderson PN.Axonal guidance molecules and the failure of axonal regeneration in the adult mammalian spinal cord. Restor Neurol Neurosci,2008;26(2-3):117-130
17. Gallo G. RhoA-kinase coordinates F-actin organization and myosin Ⅱ activity during semaphorin-3A-induced axon retraction. J Cell Sci, 2006,119(Pt 16):3413-3423
18. Fabes J, Anderson P, Brennan C, et al.Regeneration-enhancing effects of EphA4 blocking peptide following corticospinal tract injury in adult rat spinal cord. Eur J Neurosci,2007,26(9):2496-2505
19. Benson MD, Romero MI, Lush ME, et al.Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc Natl Acad Sci USA,2005,102(30): 10694-10709
20. Sayas C.L,Ariaens A,Ponsioen B,et al.GSK-3 is activiated by the tyrosine kinase Pyk2 during LPA1-mediated neurite restraction.Molecular Biology of the Cell,2006,17(4):1834-1844
21. Conrad S, Schluesener HJ, Trautmann K, etal.Prolonged lesional expression of RhoA and RhoB following spinal cord injury. J Comp Neurol,2005 487(2):166-75.
22. Duffy P, Schmandke A, Schmandke A,et al.Rho-associated kinase Ⅱ (ROCKⅡ) limits axonal growth after trauma within the adult mouse spinal cord. J Neurosci,2009,29(48):15266-15276
23. Wood-Kaczmar A, Kraus M, Ishiguro K,et al.An alternatively spliced form of glycogen synthase kinase-3β is targeted to growing neurites and growth cones. Mol Cell Neurosci,2009,42(3):184-194
24. Dill J, Wang H, Zhou F,et al.Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS. J Neurosci.2008,28(36): 8914-892
25. Trivedi N, Marsh P, Goold RG,et al.Glycogen synthase kinase-3p phosphorylation of MAP IB at Ser1260 and Thr1265 is spatially restricted to growing axons.J Cell Sci,2005,118(Pt 5):993-1005