人胎儿脑室区神经前体细胞的分离纯化、培养及鉴定

目的建立有效的人胎儿神经前体细胞分离及纯化系统。方法取人自然流产胎儿脑室区(VZ)并制备单细胞悬液,采用免疫磁珠法分离CD133阳性及CD133阴性细胞群,体外培养并比较两者增殖能力。用免疫荧光化学方法检测CD133阳性细胞群中神经上皮干细胞蛋白(nestin)的表达及诱导分化后的多向分化潜能。结果纯化后CD133阳性细胞群体外扩增能力强,在无血清培养体系中能形成神经球并可连续传代,免疫荧光化学法检测表明其nestin抗原阳性,诱导分化后可分化为神经元、星形胶质细胞及少突胶质细胞。而CD133阴性细胞在培养体系中多呈单个分散状态,神经球形成数目与CD133阳性细胞有显著性差异(P<0.05)。结论免疫磁珠法可有效分离人胎儿脑内CD133阳性细胞,且该细胞在体外培养体系中具有神经前体细胞的特征。

ISOLATION, PURIFICATION AND IDENTIFICATION OF NEURAL PRECURSORS FROM VENTRICULAR ZONE OF HUMAN FETAL BRAIN

Objective To establish an effective system of isolating and purifying neural precursors from human fetal brain. Methods The single cell suspension from the ventricular zone of human fetus was prepared and incubated with PE labeled anti-CD133 antibody. The labeled cells were subsequently incubated with anti-PE magnetic beads and separated through magnetic cell sorting system(MACS). Isolated CD133 positive and negative populations were cultured in vitro and the abilities of proliferation between them were compared. The expression of nestin antigen as well as the multipotent capacity of CD133 positive population was deected by immunofluorescent stain. Results CD133 positive cells from human fetal brain could proliferate and form typical neurospheres in serum-free culture conditions. They expressed nestin antigen, and could differentiate into both neurons and glia. On the other hand, CD133 negative cells proliferated poorly in the same culture conditions and generate significantly less neurospheres than CD133 positive cells population(P<0.05).Conclusion CD133 positive cells could be separated effectively from human fetal brain with immunomagnetic beads, and they were shown to have capacities of self-renewing and multipotential differentiation.;