大鼠胚胎神经干细胞的纯化、诱导分化及鉴定

探讨胚胎神经干细胞 (Neural stem cell,NSCs)的体外纯化扩增、保存标记、诱导分化及其鉴定方法。将14 .5 d胎龄大鼠大脑额叶皮质 NSCs在无血清 DMEM/ F12 (含 2 0 ng/ ml b FGF,2 0 ng/ m l EGF及 B2 7辅助培养液 )培养 ,利用有限稀释法将悬浮生长的单个细胞所形成的克隆球挑选出来、通过亚克隆连续传代大量扩增而纯化 ,免疫组化鉴定 nestin抗原阳性 ;选取部分 NSCs冻存、复苏后 nestin抗原阳性 ;用 Brd U孵育 NSCs,被 Brd U标记的 NSCs及其血清诱导分化后仍均呈 Brd U阳性。用血清或饲养层诱导 NSCs分化为大量表达 Tubulin- (神经元特异性抗原微管蛋白 3)阳性的神经元和 GFAP(神经胶质纤维酸性蛋白 )阳性的神经胶质细胞。由该实验可知有限稀释单细胞克隆连续传代是分离纯化、大量扩增胚胎期大脑 NSCs的简单有效方法。饲养层细胞也能诱导 NSCs分化为神经细胞。 Brd U可标记、示踪神经系统疾病动物模型 NSCs的实验治疗。掌握 NSCs的体外纯化培养、保存标记、诱导分化及鉴定方法可为进一步研究 NSCs的生物学特性及神经系统疾病的治疗提供新方法。

Purification,Induced Differentiation and Identification of Rat Embryonic Neural Stem Cells

The methods of purification, expanding, marking, conservation, induced differentiation and identification of neural stem cells (NSCs) in vitro were explored for further research and treatment of tethered cord syndrome in children and other neural system diseases. The cells derived from the cerebral cortex of frontal lobe in 14.5 d rat embryos were maintained in serum-free DMEM/F12 medium containing 20 ng/ml bFGF, 20 ng/ml EGF and B27 supplement. The NSCs of suspending single-cell-colon were isolated and passaged, were purified by limited dilution, and were expanded numerously with sub-colon in consecutive generations. Then, Nestin antigen expression was detected by immunohistochemistry techniques. The cells of the purified and expanded NSCs were frozen, recovered and incubated in BrdU, and the NSCs were induced to differentiate in serum or feeder layer. These revived NSCs from frozen cells could express Nestin antigen and could be induced in serum or feeder layer to differentiate into neurons and glias expressing tubulin-III and GFAP respectively. It is good and simple for purification and proliferation of NSCs numerously by the limited dilution and consecutive generations suspending single-cell-colon of NSCs from the cerebral cortex of frontal lobe in rat embryos. The NSCs could be induced on feeder layer to differentiate into neural cells numerously. BrdU can mark and trace NSCs for the research and treatment of the animal model of neural system diseases. By good command of the technlogies for the purification proliferation, and induced differentiation of NSCs in vitro, it is possible to find a new way for further research of the biologic specificity and the treatment of the disease in nervous system.