胶质瘤C6肿瘤干细胞蛋白质组学初步分析

Proteomics analysis of BTSC derived from C6 glioma cell line

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 analyze differences between BTSC and differentiated glioma C6 cells by method of proteomics.Methods： C6 glioma cell line was involved in this study.Cells cultures were performed under the condition of NSC.The BTSCs were identified by immunocytochemical staining of neurospheres by detecting the types of molecular markers of nestin,NSE,GFAP,and also by differentiation tests.2D-PAGE was performed to separate the proteins of the BTSC and non-BTSC.The differential protein spots were analyzed by software analysis.Results： Classical neurospheres formed in vitro by normal NSC were detected in C6 glioma cell line.The neurospheres could proliferate and increase in their diameters.For most neurospheres contained many cells expressing the NSC marker nestin and relatively few cells expressing the neuronal marker NSE or the astrocytic marker GFAP.We then assessed neurosphere cells multipotency by determining the ability to generate neurons,astrocytus,and oligodendrocytes.The neurosphere cells underwent terminal differentiation upon removal of mitogen to the medium,and all of them differentiated into GFAP-positive astrocyte-like cells but also into NSE-positive neuron-like cells.There were 84 differentially expressed protein spots observed in comparing between BTSCs and non-BTSCs by 2D-PAGE（P0.05）.Spots with 2-fold expression changes were 50,and 3-fold were 17,and 4-fold were 6.Fifty-five spots were increased,while 29 spots were decreased in BTSCs.Conclusion： There were brain tumor stem cells in C6 glioma cell line,and significant differences between BTSCs and differentiated cells.