大鼠神经干细胞分离培养及基因转染研究

Isolation, culture and transfection of neural stem cells from fetal rat telencephalon

目的 建立基因工程化神经干细胞以便为生物医学应用提供材料。方法 ①分离、培养胚鼠端脑神经干细胞 ,并用巢素 (Nestin)免疫组化染色鉴定 ;诱导神经干细胞分化 ,用神经丝 2 0 0 (NF 2 0 0 )、胶质纤维酸性蛋白 (GFAP)免疫组化染色鉴定分化细胞。②用含有脑源性神经营养因子 (BDNF)的复制缺陷型腺病毒感染神经干细胞 ,并用原位杂交检测。结果 ①获得了大量未分化、呈巢状悬浮生长的神经干细胞团 ,且能分化为神经元和神经胶质细胞。②约 80 %～ 90 %神经干细胞能被腺病毒感染 ,且经传代培养 1 2代后仍具干细胞特性。结论 从胚鼠端脑分离培养的细胞具有自我更新能力和多潜能分化能力 ,为中枢神经系统的干细胞。经基因工程化及传代后 ,仍具干细胞特性 ,可作为基础研究及神经移植供体 。

Objective To establish the genetically engineered neural stem cells(NSCs) for further biomedical applications. Methods ① After isolated from fetal rat telencephalon and identified with nestin immunocytochemical staining, NSCs were induced to differentiate. The differentiated cells were detected respectively with neurofinament (NF 200) and glial fibrillary acidic protein(GFAP) immunocytochemical staining. ② Recombinant brain-derived neurotrophic factor (BDNF) adenovirus was used to infect the obtained cells and the results were identified with in situ hybridization. Results ① Nest like clusters of neural stem cells were obtained in the suspension and the cells could be differentiated into neurons and astrocytes . ② About 80%-90% of NSCs were infected by recombinant BDNF adenovirus, which retaining the main characteristics of NSCs after 12 passages of culture. Conclusion The cells from fetal rat telencephalon possess multipotency and self renew ability and is believed to be BSCs of the central nervous system.Since they retain their characteristics and differentiation ability after passages and genetic engineering, they can be applied widely in basic research and as grafts in neural transplantation.