新生Wistar大鼠海马神经细胞的体外培养与鉴定

Identification and Culture of Neural Stem Cells Isolated from Postnatal Wistar Rat Hippocampus

为探讨原代Wistar大鼠海马神经细胞的体外培养方法,本研究取新生24h内的Wistar大鼠海马组织,无菌剪碎后采用胰酶消化法分离细胞,用含20%胎牛血清的DMEM/F12培养基培养,逐日在倒置相差显微镜下观察。结果发现,从海马组织中分离出的神经细胞具有增殖能力,细胞对数生长期为2～8d,最长培养30d;细胞经免疫荧光鉴定Nestin表达呈阳性;免疫组织化学结果显示,在传代培养细胞的胞体和突起均有NF阳性标记物,GFAP抗体和CD68抗体显色均为阴性。由此可见,分离培养的细胞是具有自我更新增殖和多分化潜能的神经元细胞。

To establish a method to isolate culture and identify the neural cells from postnatal Wistar rat hippocampus. The neural stem cells were isolated from postnatal 24 h Wistar rat hippocampus and cultured with DMEM containing 20% fetal bo- vine serum/F12 medium, and observation in inverted phase contrast microscope daily. The results showed that the neural stem cells isolated from postnatal Wistar rat hippocampus had reproductive activity and the exponential growth phase of neural stem cells was 2 to 8 d and maintain 30 d. The ceils were identified by immunofluorescence expression of Nestin positive, and immunohistochemistry results showed that the cell bodies in the subculture and processes were NF-positive markers, GFAP antibody and CD68 antibody color were negative. Thus, the cells isolated and cultured from postnatal rat hippocampus had shown a strong ability of self-renewal proliferation and multipotency of differentiation which were evidently identified as the neural stem cells.