【摘要】 目的探讨绿色荧光蛋白(green fluorescent protein,GFP)转基因大鼠胚胎脊髓神经干细胞(neural stem cells,NSC)体外增殖、分化的生物学活性以及植入体内后存活、分化和迁移的情况。方法对 GFP 转基因大鼠的胚胎脊髓 NSC 进行体外分离、培养和诱导分化,并应用特异性抗体分别对神经干细胞、神经元、星形胶质细胞和少突胶质细胞进行免疫荧光染色鉴定。建立 F344大鼠胫神经切断的动物模型,将体外稳定传代的 GFP-NSC 单细胞悬液移植于胫神经远侧段。移植12周后取材,应用激光共聚焦显微镜和普通荧光显微镜观察神经干细胞体内的存活、分化和迁移情况,并对胫神经冰冻切片行神经元特异性免疫荧光染色鉴定。结果通过体外分离培养的方法获得了稳定传代的 GFP-NSC,体外诱导分化形成神经元、星形胶质细胞和少突胶质细胞。GFP-NSC 体内移植后,部分分化为神经元,并发出轴突样的结构向远端生长。结论 GFP-NSC 具有良好的生物学活性,体内移植后可以分化为神经元,为进一步研究其体内移植防治骨骼肌失神经萎缩及治疗其他神经元损伤性疾病提供了实验依据。更多还原

【Abstract】 Objective To investigate the in vitro proliferation and differentiation of neural stem cells isolated from spinal cord of GFP transgenic rat embryos and their ability to survive,differentiate and migrate after transplantation in vivo.Methods Neural stem cells isolated from spinal cord of P14-16 pups of GFP transgenic rats were cultured and induced to differentiate in vitro.Indirect immunofluorescent cytochemistry using marker of Nestin for neural stem cells,β-tubulinⅢ for neurons,GFAP for astrocytes and NG2 for oligodendrocytes was used to identify the neural stem cells and the differentiated cells.Tibial nerve transection model was established in F344 rats and the suspension of GFP-NSCs was injected into the distal stump of the fibial nerve.Twelve weeks after stem cell transplantation,the distal part of the tibial nerve was harvested.Survival and differentiation of the transplanted GFP-NSCs were observed with confocal microscope and fluorescent microscope and the implanted part of tibial nerve was identified for neurons with β-tubulin Ⅲ by means of indirect immunofluorescent staining. Results Stable passages of GFP-NSCs could be obtained from mechanical dissociation of spinal cord of GFP transgenic rat embryos.They could differentiate into 3 kinds of neural cells under in vitro induction:neurons, astrecytes and oligodendrocytes.The GFP-NSCs could survive after being transplanted in vivo and some of them could be induced to differentiate into neurons,which sent out axons towards the denervated muscle.Conclusion Neural stem cells have good biologic activity.They can differentiate into neurons after being transplauted in vivo.This provides experimental evidence that it might be possible to delay the atrophy of denervated skeletal muscle and treat some neuron diseases by means of neural stem cell transplantation.更多还原