Rieske Piotr, Golanska Ewa, Zakrzewska Magdalena, Piaskowski Sylwester, Hulas-Bigoszewska Krystyna, Wolańczyk Magdalena, Szybka Malgorzata, Witusik-Perkowska Monika, Jaskolski Dariusz J, Zakrzewski Krzysztof, Biernat Wojciech, Krynska Barbara, Liberski Pawel P
Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Lodz, Poland.
BMC Cancer. 2009 Feb 14;9:54. doi: 10.1186/1471-2407-9-54.
Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed.
Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed.
In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum.
Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.
尽管已有报道称胶质母细胞瘤细胞培养物中存在可变分化特征，但尚未对正常神经胶质纤维酸性蛋白（GFAP）阳性祖细胞与胶质母细胞瘤细胞所表现出的分化特性进行比较分析。
采用以下方法比较胶质母细胞瘤细胞和GFAP+神经祖细胞（NHA）：暴露于神经分化培养基、脂肪生成和成骨培养基、蛋白质免疫印迹分析、免疫细胞化学、单细胞分析、溴脱氧尿苷（BrdU）掺入分析。为了表征胶质母细胞瘤细胞，进行了表皮生长因子受体（EGFR）扩增分析、杂合性缺失/微卫星不稳定性（LOH/MSI）分析和P53核苷酸序列分析。
将源自8例胶质母细胞瘤的癌细胞的体外分化与GFAP阳性正常神经祖细胞（GFAP+NNP）进行了比较。在暴露于分化培养基之前，两种类型的细胞均表现出相似的多谱系表型（CD44+/微管相关蛋白2（MAP2）+/GFAP+/波形蛋白+/βIII微管蛋白+/纤连蛋白+），并且性别决定区Y框蛋白2（SOX-2）和巢蛋白呈阳性。与GFAP+NNP相反，在从胶质母细胞瘤分离的所有细胞系中均观察到有效的分化停滞。然而，从4例胶质母细胞瘤分离的细胞亚群在血清饥饿后以不同效率分化为与GFAP+NNP的神经衍生物无法区分的衍生物。此外，源自大多数胶质母细胞瘤（8例中的7例）的细胞以及GFAP+NNP在暴露于含血清的培养基时表现出间充质分化特征。
我们的结果表明，胶质母细胞瘤细胞多谱系标志物的稳定共表达是由分化停滞导致的。根据我们的数据，高达95%的胶质母细胞瘤细胞可呈现体外多谱系表型。胶质母细胞瘤细胞的间充质分化程度较高，与正常神经祖细胞GFAP+NNP的间充质分化相似。
