人工合成多肽FGL和IKVAV促进损伤脊髓功能恢复的实验研究
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摘要
研究目的通过体外和体内两种方式探讨多肽FGL和IKVAV对脊髓损伤后恢复的影响。研究方法设计并人工合成FGL和IKVAV多肽和对照肽。1①培养神经细胞模型PC-12细胞,分为对照组和实验组,实验组中加入FGL多肽溶液。对照组预先用多聚赖氨酸包被培养板。分别于培养1、3、5、7、9d后采用细胞增殖检测CCK-8法检测两组细胞增殖情况。②取PC-12细胞。分为三组,正常对照组不作处理,损伤对照组加入H2O2刺激16h。实验组先加入FGL多肽预孵育30 min,再加H2O2刺激16h。流式细胞仪检测两组细胞凋亡情况；荧光定量PCR法检测PC-12中的核因子NF-κB mRNA。2培养星形性胶质细胞,将细胞分为对照组和实验组。对照组正常培养,实验组加入IKVAV人工合成多肽,检测两组细胞存活情况。②制作细胞划伤模型,分为正常组、对照组、实验组,正常组不作任何处理,对照组做划伤处理,实验组加入IKVAV培养一天后,制作划伤模型,三天后提取各组细胞mRNA,荧光定量PCR检测GDNFmRNA表达变化。3方法将45只大鼠随机分成三组,A组为假手术组,B组为多肽FGL和IKVAV对照肽组,C组为多肽FGL和IKVAV干预组,采用原位杂交和Western blotting免疫印迹分析脊髓损伤后硫酸软骨素蛋白多糖(CSPGS) NG2的表达；采用BBB评分法对大鼠瘫痪后肢进行运动功能评估。研究结果1实验组培养1、3、5、7、9d PC-12增殖数量均高于对照组。H2O2处理后,实验组PC-12凋亡数量及其中NF-κB mRNA的表达量均低于对照组。2 IKVAV多肽组可存活率达95%以上,实验组和对照组细胞活性没有明显差异,在划伤实验中,实验组细胞GDNF表达明显高于对照。3原位杂交和Western blotting结果显示假A组CSPGS NG2表达较弱,B组CSPGS NG2显著增多,C组给予多肽FGL干预后CSPGs NG2表达明显减弱,Western blotting结果验证了瘫痪后肢运动明显改善(均P<0.05)。结论1多肽FGL可促进PC-12细胞增殖,并抑制其凋亡2人工合成多肽IKVAV对细胞活性没有影响,可促进划伤星形胶质细胞分泌神经营养因GDNF。3多肽FGL和IKVAV可以通过减少CSPGS NG2表达促进脊髓损伤大鼠后肢运动功能恢复。
Objective:Effect of FGL and IKVAV peptide on spinal cord injury recover in vivo and vivtro.
Methods:(?)Cultured tne neural ce(?) mo(?)e. P(?) cells.THe P(?) cells. were divided into control and experimental groups. the experimental group added FGL peptide solution. Control group was pre-coated with poly-lysine plates. Cell proliferation was detected 1,3,5,7,9 d respectively by CCK-8②The PC-12 cells was divided into three groups, Damage control group added in H2O2 and irritated it for 16 hours. Take cells without stimulation as the normal control.The experimental group added in FGL peptide and H2O2 to irritate it for 16 hours.Apoptosis were detected by flow cytometry; The expression level of gene NF-κB was detected by fluorescence quantitative PCR.2. We synthesized IKVAV Peptides①cultured astroyte cells and the cells were divided into control and experimental groups. The experimental group added IKVAV synthetic peptides and the control group was added nothing.Three days later.The two groups were added the calcein-am/pi to detect the cell survival.②The cell were divided into normal group, control group, experimental group.Normal group was untreated.Control group did scratch.The experimental group was added IKVAV and made the scratch model.The GDNF mRNA level in astroyte was determined by real-time fluorescent quantitativePCR three days after.3.Forty-five Wistar rats were randomly divided 3 equal group:A group was sham operation group,B group undergoing SCI and injection of control peptide,C group undergoing SCI and injection of FGL peptide 30min after the operation. Paralyzed hind limb motor function were assessed with BBB score in rats.
Reasult:Experimental group were higher number than the control group in the cell proliferation experiments. NF-κB mRNA expression and the number of PC-12 apoptosis in the experimental group were lower than the control group 2 Both the experimental group and control group were more than 95% survival rate. In the scratch experiments, GDNF expression in the experimental group was significantly higher than the control cells 3 In situ hybridization and Western blotting showed that expression of the CSPGS NG2 was very weak in the group A,were significently increased in the group B,and were significantly down-regulated in the group C compared with those of the group B.
Conclusion:FGL peptide could promote the pc-12 cell proliferation and inhibit the apoptosis of the PC-12 cells.2 Synthetic peptide did not affect activity of cells IKVAV.The scratch injury on astoryte directly upregulated GDNFmRNA expression,and synthetic IKVAV can promote the response.3 The FGL peptide can promote the recovery of the locomotion function of the paralyzed hindlimbs by inhibinting the regulating of CSPGs NG2.
Objective:Effect of FGL and IKVAV peptide on spinal cord injury recover in vivo and vivtro.
Methods:(?)Cultured tne neural ce(?) mo(?)e. P(?) cells.THe P(?) cells. were divided into control and experimental groups. the experimental group added FGL peptide solution. Control group was pre-coated with poly-lysine plates. Cell proliferation was detected 1,3,5,7,9 d respectively by CCK-8②The PC-12 cells was divided into three groups, Damage control group added in H2O2 and irritated it for 16 hours. Take cells without stimulation as the normal control.The experimental group added in FGL peptide and H2O2 to irritate it for 16 hours.Apoptosis were detected by flow cytometry; The expression level of gene NF-κB was detected by fluorescence quantitative PCR.2. We synthesized IKVAV Peptides①cultured astroyte cells and the cells were divided into control and experimental groups. The experimental group added IKVAV synthetic peptides and the control group was added nothing.Three days later.The two groups were added the calcein-am/pi to detect the cell survival.②The cell were divided into normal group, control group, experimental group.Normal group was untreated.Control group did scratch.The experimental group was added IKVAV and made the scratch model.The GDNF mRNA level in astroyte was determined by real-time fluorescent quantitativePCR three days after.3.Forty-five Wistar rats were randomly divided 3 equal group:A group was sham operation group,B group undergoing SCI and injection of control peptide,C group undergoing SCI and injection of FGL peptide 30min after the operation. Paralyzed hind limb motor function were assessed with BBB score in rats.
Reasult:Experimental group were higher number than the control group in the cell proliferation experiments. NF-κB mRNA expression and the number of PC-12 apoptosis in the experimental group were lower than the control group 2 Both the experimental group and control group were more than 95% survival rate. In the scratch experiments, GDNF expression in the experimental group was significantly higher than the control cells 3 In situ hybridization and Western blotting showed that expression of the CSPGS NG2 was very weak in the group A,were significently increased in the group B,and were significantly down-regulated in the group C compared with those of the group B.
Conclusion:FGL peptide could promote the pc-12 cell proliferation and inhibit the apoptosis of the PC-12 cells.2 Synthetic peptide did not affect activity of cells IKVAV.The scratch injury on astoryte directly upregulated GDNFmRNA expression,and synthetic IKVAV can promote the response.3 The FGL peptide can promote the recovery of the locomotion function of the paralyzed hindlimbs by inhibinting the regulating of CSPGs NG2.
引文
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[2](?)(?)orl. Po(?)(?)v. Nik(?)(?)(?) Mesyede(?). Igor(?). Krae(?). et al. A cell adhesion molecule mimetic, FGL peptide, induces tne dentate gyrus of aged rats:ε three-dimensional ultra structural study[J]. Euro-pean Journal of Neuro science,2008,27(6):301-304.
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[5]Jiang B, liu JH, BaoYM, et al. Hydmgen peroxide-induced apoptosis in PC-12 cell the effect of protective of puerarin[J]. Cell Bio Int,2003, (12):1025-1031.
[6]Dasari VR, Veeravalli KK, et al. Neuronal apoptosis is inhibited by cord bllood stem cells after spinal cord injury[J]. Neurotrauma,2009,26(11):2057-2069.
[7]张钦,吴继彬,黄晨等罗格列酮对大鼠脊髓损伤后神经细胞凋亡的影响[J].山东医药,2010,50(22)：35-36。
[8]Ronn, L. C., Hartz, B. P, Bock, E. The neural cell adhesion molecule(NCAM) indevelopment and plasticity of the nervous system[J].EXP Gerontol,1998,33(3):853-864.
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[12]Schneider A., Martin-Villalba A., Weih, F., et al. NF-kappaB is activated and promotes cell death in focal cerebral ischemia[J]. Nat. Med,1999,5(9):554-559.
[13]陈吉相,季艳梅,孙圣刚等依达拉奉对脑出血大鼠细胞凋亡及NF-κB表达的影响[J].山东医药,2007,47(1)：7-8。
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