Redies C, Lendahl U, McKay R D
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge.
J Neurosci Res. 1991 Dec;30(4):601-15. doi: 10.1002/jnr.490300403.
Recently, various techniques have been developed to transfer oncogenes into brain cells in order to generate immortalized neural cell lines. It is of interest to establish how well such cell lines reflect their cellular origin. Here we report the characterization of sixteen cell lines from mouse cerebellum and, as a control, six cell lines from skin. Lines were established by immortalizing postnatal primary cell cultures with a retrovirus carrying a modified temperature-sensitive variant of SV40 large T antigen. The cell lines reflect many properties of the cell type from which they were derived. All of the sixteen cerebellar lines expressed one or more markers of the neural precursor cells, namely, nestin and epitopes for NG2 and A2B5. In contrast, none of the six skin lines expressed neural precursor markers. Both types of cell lines expressed vimentin and fibronectin. Differentiation occurred in some of the cerebellar lines and was enhanced in defined medium. A small percentage of cerebellar cells, usually less than 5%, was positive for a marker of differentiation, e.g., glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), or L1. Expression of GFAP colocalized with that of nestin at varying levels of intensity, indicating a gradual replacement of nestin by GFAP in the cytoskeleton. Both the cells positive for precursor markers and those positive for differentiation markers tended to be located in clusters, suggesting that stochastic processes or cell-cell interactions are important for the determination of the fate of cells within a clonal cell line in vitro. The degree of differentiation seemed to correlate with a shift from serum-containing to defined medium, but not with a shift from the permissive to the nonpermissive temperature for T antigen expression. The immortalization approach described here thus allows the establishment of cell lines which are "captured" in the precursor state of the developing mouse neuroepithelium.
最近，已开发出各种技术将癌基因导入脑细胞，以生成永生化神经细胞系。确定此类细胞系在多大程度上反映其细胞起源很有意义。在此，我们报告了来自小鼠小脑的16个细胞系的特征，并作为对照，报告了来自皮肤的6个细胞系的特征。通过用携带SV40大T抗原修饰的温度敏感变体的逆转录病毒使出生后原代细胞培养物永生化来建立细胞系。这些细胞系反映了它们所源自的细胞类型的许多特性。16个小脑细胞系均表达一种或多种神经前体细胞标志物，即巢蛋白以及NG2和A2B5的表位。相比之下，6个皮肤细胞系均未表达神经前体标志物。两种类型的细胞系均表达波形蛋白和纤连蛋白。一些小脑细胞系发生了分化，在限定培养基中分化增强。一小部分小脑细胞（通常少于5%）对分化标志物呈阳性，例如胶质纤维酸性蛋白（GFAP）、半乳糖脑苷脂（GalC）或L1。GFAP的表达与巢蛋白的表达在不同强度水平上共定位，表明在细胞骨架中巢蛋白逐渐被GFAP取代。前体标志物阳性的细胞和分化标志物阳性的细胞往往都聚集成簇，这表明随机过程或细胞间相互作用对于体外克隆细胞系内细胞命运的决定很重要。分化程度似乎与从含血清培养基转变为限定培养基相关，但与T抗原表达从允许温度转变为非允许温度无关。因此，这里描述的永生化方法允许建立在发育中的小鼠神经上皮前体状态下“捕获”的细胞系。
