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目的:运用蛋白质组学的方法,对C6脑肿瘤干细胞(BTSC)和非BTSC细胞进行配对研究,建立差异性蛋白文库,为探讨胶质瘤病因提供初步线索。方法:在C6细胞中,参照神经干细胞(NSC)培养条件进行培养。出现典型的神经球后,利用免疫荧光染色检测细胞标记,包括nestin,神经元特异性烯醇化酶(NSE),神经胶质细胞纤维酸性蛋白(GFAP)等,以及诱导分化等方法,鉴定神经球细胞是否为BTSC。收集神经球和非神经球细胞,进行配对,利用双向凝胶电泳(2D-PAGE)检测细胞总体蛋白,分析差异性蛋白点。结果:C6细胞在NSC培养条件下,出现比较典型的神经球。神经球细胞nestin阳性,NSE和GFAP阴性。经诱导发生分化,分化后细胞形态与C6相似,能够表达NSE和GFAP等多种表型细胞标记,证实这种神经球细胞为肿瘤干细胞。在2D-PAGE结果中,C6肿瘤干细胞和非干细胞配对比较,有统计学差异(P<0.05)的蛋白点共有84个,大于2倍差异蛋白点有50个,大于3倍的有17个,大于4倍的有6个,最大差异倍数为11.607 2。在BTSC中,有55个差异蛋白表达增多(上调),29个蛋白表达下降(下调)。结论:胶质瘤C6细胞系中存在肿瘤干细胞,在蛋白质组学方面与非BTSC细胞有明显差异。
OBJECTIVE: To perform a paired study on C6 brain tumor stem cells (BTSC) and non-BTSC cells using proteomics methods and to establish a differential protein library to provide preliminary clues for exploring the etiology of glioma. Methods: C6 cells were cultured according to the culture conditions of neural stem cells (NSCs). After the appearance of typical neurospheres, we used immunofluorescent staining to detect the cell markers, including nestin, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and the induction of differentiation Whether the cell is BTSC. Neurospheres and non-neurosphere cells were collected and paired, and the total cellular protein was detected by two-dimensional gel electrophoresis (2D-PAGE), and the differential protein spots were analyzed. Results: C6 cells showed typical neurospheres under NSC culture conditions. Neurospheres nestin positive, NSE and GFAP negative. After induction of differentiation, differentiation and cell morphology similar to C6, NSE and GFAP can express a variety of phenotypic cell markers, confirmed that such neurospheres as cancer stem cells. In 2D-PAGE, there were 84 protein spots with statistically significant differences (P <0.05), 50 more than 2 times more protein spots, 17 more than 3 times more spots, There are 6 more than 4 times and the maximum multiple of difference is 11.607 2. In BTSC, 55 differentially expressed proteins were upregulated (upregulated) and 29 proteins were downregulated (downregulated). Conclusion: There are tumor stem cells in glioma C6 cell line, which have obvious difference in proteomics with non-BTSC cells.