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采用蔗糖密度梯度超速离心法分离纯化高尔基体,双向凝胶电泳(2-DE)分离高尔基体蛋白质,用ImageMaster 2D软件分析所得图谱,基质辅助激光解吸离子化飞行时间质谱(MALDI—TOF MS)鉴定蛋白质点等一系列亚细胞器蛋白质组学方法建立胃癌细胞内高尔基体的蛋白图谱。结果显示分离出的纯度较高的高尔基体建立了分辨率和重复性均较好的双向电泳图谱,运用质谱技术鉴定出12个蛋白质,包括蛋白合成相关蛋白、膜融合蛋白、调节蛋白、凋亡相关蛋白、运输蛋白、细胞增殖分化相关蛋白。通过亚细胞器分离纯化,双向电泳的蛋白分离及MALDI-TOF MS蛋白鉴定分析,首次成功建立了胃癌细胞SGC7901中高尔基体的蛋白质组学技术路线,为胃癌细胞内高尔基体功能的深入研究奠定了基础。
The Golgi apparatus was isolated and purified by sucrose density gradient ultracentrifugation. The Golgi apparatus was separated by two-dimensional gel electrophoresis (2-DE) and analyzed by ImageMaster 2D software. MALDI-TOF MS Protein spots and a series of subcellular organelle proteomics methods to establish the protein profile of gastric Golgi in gastric cancer cells. The results showed that the isolated high purity Golgi apparatus established a two-dimensional gel electrophoresis pattern with good resolution and repeatability. The 12 proteins identified by mass spectrometry, including protein synthesis related proteins, membrane fusion proteins, regulatory proteins, apoptosis Related proteins, transport proteins, cell proliferation and differentiation related proteins. Through subcellular organelle purification, two-dimensional electrophoresis protein separation and MALDI-TOF MS protein identification and analysis, the first successful establishment of human gastric cancer cell SGC7901 Golgi proteomics technology line for gastric cancer cells in-depth study of Golgi apparatus laid the foundation .