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目的建立有效的人胚胎神经干/祖细胞分离及纯化方法以达到临床需要。方法无菌取胎脑室管膜下区组织,经反复机械吹打制备细胞混悬液,采用贴壁法进行体外培养、传代。传代后用Nestin进行细胞鉴定。结果应用贴壁法进行胎脑室管膜下区神经干/祖细胞的培养能够稳定传代20代以上;共聚焦显微镜下可见Nestin阳性细胞表达呈递增趋势,传至P3(passage3)阳性率为35%,P7为79%,P12为90%,P15为99%。传到前七代时分别做细胞存活率鉴定,可达(82.57±1.38)%。结论成功建立了来源于人胚室下区的神经干/祖细胞的贴壁法培养,为该细胞移植治疗神经系统疾病提供了技术方法支持。
Objective To establish an effective method of human embryonic neural stem / progenitor cells isolation and purification in order to achieve clinical needs. Methods The subventricular zone tissue of fetal brain was aseptically removed. The cell suspension was prepared by repeated mechanical beating. The cells were cultured and passaged by adherence method. After passage, Nestin was used for cell identification. Results The adhesion of neural stem / progenitor cells in fetal subependymal zone could be stably passaged for 20 generations or more. The expression of Nestin positive cells showed an increasing trend under the confocal microscope, and the positive rate of P3 (passage 3) was 35% 79% for P7, 90% for P12 and 99% for P15. Differentiated to the first seven generations of cell survival were identified, up to (82.57 ± 1.38)%. Conclusion The successful adherent culture of neural stem / progenitor cells derived from the subventricular zone of human embryo has been established, which provides technical support for the treatment of neurological diseases by cell transplantation.