胎鼠大脑皮质神经干细胞的分离培养和鉴定:有向多种成体神经干细胞分化的潜能吗?

Preparation,cultivation and identification of neural stem cells in fetal rat cerebral cortex:Do they have multiple differentiation potency?

背景：脑室周围白质软化是早产儿脑损伤的主要类型,迄今尚无防治方法。对丢失大量少突胶质细胞的白质进行神经干细胞移植,理论上应是治疗脑室周围白质软化最为理想的方案。目的：体外培养制备具有多向分化潜能的胎鼠神经干细胞,以供后期实验经脑室移植应用。方法：取孕12~14d胎鼠大脑皮质组织,剪成1.0mm3小块制备单细胞悬液分离纯化,待形成细胞球后加入含小牛血清的DMEM/F12培养基进行诱导分化培养。观察神经干细胞的原代、传代培养情况,免疫组化法对神经干细胞分化情况进行鉴定。结果与结论：培养的神经干细胞活力为（94.3±2.2）%,原代培养3d形成神经球,体外传至10代左右,神经球的细胞团增殖速度明显减慢,部分细胞老化。各代神经球均呈巢蛋白染色阳性,可确认为神经干细胞。进一步对第4代神经球诱导分化培养后,免疫组化结果分别呈GFAP,β-tublin和O4阳性。提示所制备的神经干细胞具有自我更新和增殖能力,并具备向神经元、星形胶质细胞及少突胶质细胞分化的潜能。

BACKGROUND：Periventricular leukomalacia is a major syndrome of premature infant brain injury,which has been not prevented and cured yet.Theoretically,neural stem cells which were transplanted into white matter with an absence of oligodendroglial cells might be an ideal method to cure periventricular leukomalacia.OBJECTIVE：To prepare the multi-lineage potential of neural stem cells for the use of intraventricular transplantation.METHODS：Cerebral cortex was obtained from 12-14-day fetal rats and sectioned into 1.0-mm3 sections.The single cell suspension was separated and purified.The neurospheres were incubated with DMEM/F12 culture medium containing fetal bovine serum to observe primary and passage culture of neural stem cells.The differentiation of neural stem cells was determined using immunohistochemical method.RESULTS AND CONCLUSION：The viability of cultured neural stem cells was （94.3±2.2）%.The neurosphere was formed at day 3 after primary culture.The proliferation of neurosphere slowed down after 10-passage culture,and some cells became old.All neurospheres were positively Nestin-staining,thus they were considered as neural stem cells.A further incubation of 4-passage neurospheres,immunohistochemical method indicated that the neurosphere was positively GFAP,β-tublin,and O4 staining,respectively.This suggested that cultured neural stem cells are able to self-renew,proliferate,and differentiate into neurons,astrocytes and oligodendroglial cells.