胶原/丝素蛋白支架联合神经干细胞治疗创伤性脊髓损伤

Collagen/silk fibroin scaffold combined with neural stem cells in the treatment of traumatic spinal cord injury

背景:随着交通事故、坠落伤、运动损伤等不断增多,创伤性脊髓损伤已成为危及脊髓健康的一个重要问题。目的:探讨胶原/丝素蛋白支架联合神经干细胞治疗创伤性脊髓损伤的疗效。方法:①分别提取胶原和丝素蛋白原料,将二者以质量比2∶4混合,采用真空冷冻干燥法制备胶原/丝素蛋白支架;将第3代GFP小鼠神经干细胞接种至胶原/丝素蛋白支架上,光学显微镜和扫描电镜下观察神经干细胞生长情况。②采用随机数字表达将40只成年SD大鼠分5组,正常组不进行任何处理,模型组建立T10段脊髓缺损模型,干细胞组脊髓缺损处注射神经干细胞,支架组脊髓缺损处植入胶原/丝素蛋白支架,联合组脊髓缺损处植入接种神经干细胞的胶原/丝素蛋白支架,每组8只。术后每周进行旷场实验BBB评分和斜坡实验,术后第8周行神经诱发电位检测、苏木精-伊红和免疫荧光染色,评价创伤性脊髓损伤大鼠恢复情况。结果与结论:①光学显微镜和扫描电镜下观察显示,胶原/丝素蛋白支架上有利于神经干细胞的黏附、伸展与分化。②旷场实验BBB评分和斜坡实验结果显示,脊髓损伤各组大鼠的运动功能随时间的延长均有不同程度的恢复,各治疗组大鼠的运动功能的恢复速度与程度均优于模型组,其中以联合组大鼠运动功能恢复最好。术后第8周,脊髓损伤后各治疗组大鼠的运动诱发电位和体感诱发电位的潜伏期、振幅检测结果均优于模型组(P<0.05),联合组检测结果优于干细胞组、支架组(P<0.05)。术后第8周苏木精-伊红染色显示,模型组大鼠脊髓损伤部位修复效果最差,联合组修复效果最好;免疫荧光染色显示,模型组大鼠脊髓损伤部位神经丝蛋白阳性细胞最少,各治疗组神经丝蛋白阳性细胞均多于模型组,其中以联合组最多。③胶原/丝素蛋白支架联合神经干细胞对创伤性脊髓损伤大鼠双下肢功能改善和脊髓组织修复具有一定效果。

BACKGROUND:With the increasing number of traffic accidents,fall injuries,and sports injuries,traumatic spinal cord injury has become the primary disease that threatens spinal cord health.OBJECTIVE:To investigate the efficacy of collagen/silk fibroin scaffold combined with neural stem cells in the treatment of traumatic spinal cord injury.METHODS:(1)Collagen and silk fibroin raw materials were extracted separately and mixed in a mass ratio of 2:4.Collagen/silk fibroin scaffold was prepared by vacuum freeze-drying.Passage 3 GFP mouse neural stem cells were seeded on collagen/silk fibroin scaffolds.Neural stem cell growth was observed under light microscope and scanning electron microscope.(2)Totally 40 adult SD rats were randomly divided into five groups(n=8 per group).The normal group did not receive any treatment.In the model group,a T_(10) segment spinal cord defect model was established.In the stem cell group,neural stem cells were injected into the spinal cord defect.Collagen/silk fibroin scaffolds were implanted at the spinal cord defect site in the scaffold group.Collagen/silk fibroin scaffolds seeded with neural stem cells were implanted at the spinal cord defect site of the combination group.Open field test BBB score and slope test were performed every week after operation.Evoked potential detection,hematoxylin-eosin staining and immunofluorescence staining were conducted to evaluate the recovery of traumatic spinal cord injury rats.RESULTS AND CONCLUSION:(1)Optical microscopy and scanning electron microscopy showed that the collagen/silk fibroin scaffolds were favorable for the adhesion,extension and differentiation of neural stem cells.(2)Open field test BBB score and slope test results demonstrated that the motor function of the rats in each spinal cord injury group recovered to different degrees with the prolongation of time.The speed and degree of motor function recovery of the rats in each treatment group were better than those in the model group,and the motor function recovery of the rats in the combination group was the best.At 8 weeks after operation,the detection results of latency and amplitude of motor evoked potentials and somatosensory evoked potentials of rats in each treatment group were better than those in the model group after spinal cord injury(P<0.05).The detection results of the combination group were better than those of the stem cell group and the scaffold group(P<0.05).At 8 weeks after operation,hematoxylin-eosin staining demonstrated that the repair effect of the injured spinal cord in the model group was the worst and the repair effect of the combination group was the best.Immunofluorescence staining exhibited that the number of neurofilament protein-positive cells at the spinal cord injury site of the rats in the model group was the least,and the number of neurofilament protein-positive cells in each treatment group was more than that in the model group,among which the number of the combination group was the most.(3)Collagen/silk fibroin scaffold combined with neural stem cells has a certain effect on the improvement of the function of both lower limbs and the repair of spinal cord tissue in rats with traumatic spinal cord injury.