肝癌细胞亚细胞组分的双向凝胶电泳分析

Two-dimensional gel electrophoresis of subcellular fractions of hepatoma cells

目的建立超速离心方法分离亚细胞组分的技术路线,以提高肝癌细胞蛋白质组的双向凝胶电泳分离效率。方法体外培养的肝癌细胞QGY-7703匀浆破碎后,经差速离心分离成细胞核、20000×g沉淀、100000×g沉淀和细胞浆4个亚细胞组分,分别进行双向凝胶电泳,然后用ImageMaster软件分析电泳图谱。结果亚细胞组分分离后,电泳分辨率明显提高,特别是细胞核和细胞浆蛋白质的检出效率得到很大提高。不同亚细胞蛋白质组电泳图谱的差异显示了其蛋白质组构成的不同。结论超速离心是一种有效的亚细胞组分分离手段,蛋白质组技术与超速离心技术的结合能克服全细胞双向凝胶电泳的一些缺陷,并可将蛋白质组研究引向亚细胞水平。

Objective To seek a better profiling of proteins of hepatoma cells. Methods The homogenate of hepatoma cells QGY-7703 was fractionated into four parts by differential centrifugation: the nuclei, the pellet by 20 000×g, the pellet by 100 000×g and the cytosolic supernatant. The four fractions were submitted to two-dimensional gel electrophoresis and their electrophoretic patterns were analyzed. Results In comparision with the protein pattern of hepatoma cells not fractionated, the patterns of the four fractions display many more protein spots, and a large number of proteins present in the nuclei and cytosolic supernatant were not shown in the not-fractionated samples. Conclusions Preparation of subcellular fractions before electrophoretic procedures proves to be very useful; not only can it improve the results of two-dimensional gel electrophoresis, but also can lead to research into the subcellular level.