双向电泳技术分析大鼠星形细胞和C6细胞的蛋白差异

Proteomic analysis of Astrocytes and C6 Glioma Cells

目的:建立大鼠星形胶质细胞和C6胶质瘤细胞的双向电泳(2-DE)技术体系,寻找星形胶质细胞和C6胶质瘤细胞的蛋白表达差异。方法:提取大鼠星形胶质细胞和C6胶质瘤细胞中的总蛋白,进行第一向等电聚焦(IEF)和第二向十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离总蛋白,GS-800获得凝胶图像,并测量不同凝胶间蛋白斑点在IEF和SDS-PAGE方向上的位置偏差,使用PDQuest7.3软件对凝胶图像进行分析,找出星形胶质细胞和C6胶质瘤细胞的差异表达蛋白。结果:星形胶质细胞和C6胶质瘤细胞的蛋白表达谱上,分别检测到523±16和550±20个蛋白点,平均匹配点数为452±18和472±21,匹配率达86.4%和85.8%,不同凝胶间蛋白质点在IEF方向的偏差为(0.67±0.48)mm,在SDS-PAGE方向上的偏差为(0.73±0.56)mm,对比分析了星形胶质细胞和C6胶质瘤细胞的2-DE图谱,发现有24个点发生了明显的表达变化(P<0.01),在星形胶质细胞中高表达的点有8个,在C6胶质瘤细胞中高表达的点有14个,星形胶质细胞中没有发现表达的点有2个。结论:建立了大鼠星形胶质细胞和C6胶质瘤细胞的双向电泳技术体系,发现两者的双向电泳图谱存在明显差异,C6胶质瘤细胞中表达上调以及星形胶质细胞中没有发现表达的蛋白可能与肿瘤的发生,神经干细胞的迁移有关,表达下调的蛋白可能与抑制肿瘤的发生有关。

Objective： To establish two-dimensional gel electrophoresis（2-DE） system for astrocytes and C6 glioma cells, and find differences of protein expression. Methods： By extracting the total proteins from the astrocytes and C6 glioma cells, quantitation, and then running isoelectric focusing （IEF） electrophores as the first dimension, and the sodium dodecyl sulfate polyacrylamide gel electro- pheresis （SDS-PAGE） as the second dimension to separate the total proteins, the images were abtained through the GS-800 scanning, and the position deviation of matched spots in IEF and SDS-PAGE were measured. The protein spots were analyzed by PDQuest 7.3 software and then the protein spots were found to express differently. Result： For astrocytes ,the average protein spots were 523±16, and 452±18 spots were matched with the average matching rate of 86.4%.For C6 glioma cells, the average protein spots were 550＋20, and 472＋21 spots were matched with the average matching rate of 85.8%.The average position deviation of matched spots was 0.67±0.48mm in IEF direction, and 0.73±0.56 mm in SDS-PAGE direction. 24 proteins in the C6 glioma cells groups were noted to have qualitative or quantitative differences compared with the astrocytes groups. Among the 24 proteins, the 2 proteins were unique, the 14 proteins upregulated and the 8 proteins down-regulated. Conclusions： There are distinctive differences between 2-DE maps of astro- cytes and C6 glioma cell group, downregulated proteins in C6 glioma cell may be involved in restraining tumor, and upregulated or unique proteins may participate in the formation of tumor and migration of NSCs.