含FGL活性片段的自组装多肽支架材料修复大鼠脊髓损伤的研究

Effect of functionalized peptide self-assembling nanofiber scaffolds containing FGL motif on rat spinal cord injury repair

目的 建立大鼠脊髓损伤模型,将含FGL功能化多肽自组装神经支架材料（FGL-NS）植入到脊髓损伤局部,探讨FGL-NS神经支架材料修复脊髓损伤的效果和机制.方法 麻醉大鼠后,暴露胸10水平脊髓,用特制血管夹（夹力24g）钳夹1 min,建立大鼠胸髓钳夹损伤模型.设立空白组、对照组和实验组,分别于损伤后24h,暴露脊髓损伤局部,将2.5μl等渗葡萄糖溶液、1％ RADA-16和1％ FGL-NS多肽溶液注射入损伤局部.术后3d、1、3、5、7和9周分别采用Basso-Beattie-Bresnahan（BBB）评分评估大鼠脊髓损伤经治疗后运动功能恢复情况,术后9周取脊髓损伤部位组织,行半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3、神经丝蛋白-200 （NF-200）和胶质纤维酸性蛋白（GFAP）免疫组织化学染色评估损伤部位细胞凋亡、轴突再生和瘢痕形成情况.结果 成功建立大鼠胸髓钳夹损伤模型.BBB运动学评分大鼠脊髓损伤后经FGL-NS材料治疗后,BBB评分随时间逐渐升高,自伤后第5周起,显著高于RADA-16治疗组和空白组,大鼠运动功能显著改善.免疫组织化学染色结果显示,损伤后第9周,FGL-NS治疗组脊髓损伤局部可见大量NF-200阳性的神经元细胞[（35.32±3.12）个/视野],显著高于RADA-16组[（18.56±2.64）个/视野]和空白组[（14.83±1.43）个/视野],Caspase-3阳性的凋亡细胞[（22.45 ±2.74）个/视野]显著少于RADA-16组[（30.86 ±3.75）个/视野],而且损伤区GFAP染色的积分吸光度（IA）值为0.50±0.02,明显小于对照组（1.30 ±0.09）和空白组（1.60±0.11）.结论 功能化多肽自组装神经支架材料FGL-NS能促进脊髓损伤大鼠的运动功能恢复,能减少脊髓损伤部位细胞凋亡,促进神经元再生,并减少胶质瘢痕形成.

Objective To establish a rat model of spinal cord injury （SCI） and evaluate the effect of in vivo treatment of SCI with FGL-NS peptide that could be self-assembled in vivo into functionalized nanofiber scaffold.Methods The rats were anesthetized and treated with laminectomy at the T10 vertebral segment.Then,the SCI models were established by compressing the spinal cord with a 24 g modified aneurysm clip.One day after SCI,the animals were respectively injected with 2.5 μl 580 μmol/L solution of glucose,1％ RADA-16 and 1％ FGL-NS peptide solution into the injured zone.After treatment for 3 days,1 week,3 weeks,5 weeks,7 weeks and 9 weeks,the functional recovery of SCI rats was evaluated by the Basso,Beattie and Bresnahan Locomotor Rating Scale.After treatment for 9 weeks,apoptosis rate and the number of neurons with neurite and astrocyte were detected by Caspase-3,neurofilament-200 （NF-200） and glial fibrillary acidic protein （GFAP） immunohistochemistry.Results The model of SCI in rats was established successfully.The Basso-Beattie-Bresnahan （BBB） scores of SCI rats treated with FGL-NS were higher than those in RADA-16 group after treatment for 5 weeks.The number of Caspase-3 positive cells in injuried zone treated with FGL-NS was （22.45 ± 2.74） per field,which was significantly less than in RADA-16 group [（30.86 ±3.75） per field].Meanwhile,the number of neurons with neurite in FGL-NS group was （35.32 ±3.12） per field,significantly more than that in RADA-16 group [（18.56 ±2.64） per field] and blank group [（14.83 ± 1.43） per field].Furthormore,FGL-NS inhibited the formation of astrogila at the site of injury.The integrated option density （IOD） in injuried zone treated with FGL-NS was 0.50 ± 0.02,significantly less than in control group （1.30 ± 0.09） and blank group （1.60 ± 0.11）.Conclusion Functionalized peptide self-assembling nanofiber scaffolds containing FGL motif could be a useful biomaterial in nerve tissue engineering for improving the functional recovery of SCI rats,inhibiting the formation of astrogila,reducing the cell death and increasing the number of neurons in the injured zone.