海马神经干细胞的分化特征及鉴定

Differentiational characters and identification of neural stem cells derived from hippocampus of neonatal rats

目的从新生SD大鼠海马分离、培养并鉴定神经干细胞。方法分离新生SD大鼠海马组织,经机械吹打后,加入含有20ng/mL的b-FGF以及B27的DMEM/F12无血清培养基悬浮培养具有自我更新和多向分化能力的细胞群,7d后将一部分细胞固定并采用免疫细胞化学技术检测这些细胞中神经上皮干细胞巢蛋白(Nestin)的表达;另一部分细胞加入含10%小牛血清的DMEM/F12培养基使其贴壁分化生长7d后固定并检测特异性成熟神经元抗原Tuj1以及星形胶质细胞抗原GFAP,少突胶质细胞抗原Galc的表达。结果从海马分离的细胞群具有自我更新能力,表达神经上皮干细胞特异性巢蛋白,它们分化成熟后可以表达神经元、星形胶质细胞和少突胶质细胞的特异性抗原。结论从新生SD大鼠海马分离出具有自我更新能力和多向分化潜能的神经干细胞群,它们有很强的分裂、增殖能力,是中枢神经系统的干细胞。

[Objective] To isolate and identify neural stem ceils derived from hippocampus of neonaral rats. [Methods] The ceils obtained from the hippoeampus of neonatal rats were incubated in serum-free DMEM/F12 media added with 20ng/ml b-FGF, B27. Some of them were suspension cultured in a 24-well-plate. On day 7 after incubation, some of the ceils were fixed and subjected to immunocytochemistry to detect the nestin antigen for the ceil clone. The other ceils were incubated in DMEM/F12 media added with 10% bovine serum. On day 7 after incubation the ceils were fixed and stained by using immunocytochemistry to detect the specific antigen of neuron （Tuj1）, astrocyte（GFAP）, and oligodendrocyte（Galc）. [Result] The ceils derived from the hippocampus of neonatal rats expressed nestin antigen and have the potential to form clones and differentiate into neurons, astroeytes and oligodendrocytes. [Conclusion] Muhipotent ceil line which expressed nestin antigen has been isolated and identified. These ceils showed great ability to self-renewal, supporting their character of neural stem ceils.