人胚神经干细胞移植对脊髓横切大鼠后肢运动功能恢复及脊髓再生的观察

Effect of human embryonic neural stem cells on the recovery of motor function of hindlimb and observation on the regeneration in rats with transected spinal cord

目的:观察脊髓横切大鼠人胚神经干细胞移植后功能恢复和神经干细胞的存活和分化情况,以证实其对脊髓损伤后截瘫的治疗作用。方法:从20周人胚胎分离神经干细胞进行鉴定后长期培养,培养了6个月的细胞于移植前48h标记BrdU。成年雌性Wistar大鼠于T11水平行椎板切开术并横切腰段脊髓,单纯随机分为实验组和对照组,各15只。实验组立体定向注射细胞进入两侧脊髓断端中线的4个部位(薄束、楔束、灰质联合及皮质脊髓束),每个部位注射0.5μL,细胞密度为10000个/μL,对照组注射同等量的生理盐水。利用BBB评分客观评价后肢运动功能的恢复。移植3个月后利用双标免疫组化和电子显微镜观察神经干细胞的存活和分化情况。结果:体外培养神经干细胞达到了10个月。移植神经干细胞的截瘫模型大鼠表现为后肢运动功能的逐渐恢复,3个月后BBB评分达到平均13分,对照组评分2.5分,两组差异有非常显著性意义(P<0.01)。细胞在宿主体内存活良好,它们向受伤部位迁移。电镜观察发现移植神经干细胞的大鼠在横切脊髓的部位有再生活跃的髓鞘。结论:人胚胎神经干细胞提供了一种有效的对哺乳动物脊髓损伤修复的策略,可能为脊髓损伤后再生带来新的修复手段。

AIM:To observe the functional recovery, survival and differentiation of neural stem cells(NSCs) after transplantation of human embryonic neural stem cell in rats with spinal cord transection,so as to identify its curative effect on paraplegia after spinal injury. METHODS:NSCs,separated from human fetus at about 20 weeks,were identified for long term culture.After 6 month culture,cells were marked with BrdU 48 hours before transplantation.Adult female Wistar rats were performed a sterile laminectomy at T11 and the spinal cords were transected.The rats were divided randomly into study group(n=15) and control group(n=15).Rats in the study group received sterotaxic injections of NSCs into four sites of the midline of both cord stumps(ventral funiculus,gray comissure,dorsal corticospinal tract and gracile fasculus) immediately after transection(0.5 μL for every site),and the cell density was 10 000 cells/μL,while rats in the control group were injected with saline of the same volume. Hindlimb motor function was assessed by using the Basso Beattie Bresnahan(BBB) locomotor rating scale.Three months after transplantation,their survival and differentiation were tested with double label immunohistochemistry under electron microscopy. RESULTS:Human NSCs could be cultured in vitro for 10 months. Locomotor function of the paraplegic rats was improved,and the mean BBB score reached 13, while the BBB score in the control group was 2.5,there was extremely significant difference between the two groups(P< 0.01).Transplanted cells survived for at least 3 months,migrated into the lesion.Electromicrographs indicated that the axons displayed remyelinization around the spinal cord transection site. CONCLUSION:Human NSCs transplantation provides a useful repair strategy for the mammalion animals with spinal cord injury and will bring a new therapy means for the clinical patients.