墓头回提取物作用白血病K562细胞蛋白双向电泳方法的建立  

Intervention of K562 Cell by Diversifolious Patrinia Root Extract and Establishment of the Two-Dimensional Electrophoresis Maps

作　　者：刘婷婷[1] 卫东锋[1] 赵春燕[2] 苏刚[3] 吴剑华[2] 卢艳[2] 曹慧明[2] 程卫东[1] 

机构地区：[1]兰州大学基础医学院中西医结合临床研究所,甘肃兰州730000 [2]兰州大学药学院药物化学研究所,甘肃兰州730000 [3]兰州大学基础医学院遗传研究所,甘肃兰州730000

出　　处：《时珍国医国药》2011年第8期1871-1874,共4页Lishizhen Medicine and Materia Medica Research

基　　金：甘肃省科技厅国际合作项目(No.0804WCGA126);甘肃省青年科技基金(No.099RJYA008);环境化学与生态毒理学国家重点实验室开放基金(No.KF2008-17)

摘　　要：目的建立适用于体外培养的K562细胞总蛋白的双向电泳图谱,提高电泳分辨率和重复性,以进一步了解中药墓头回作用于白血病细胞的新机制。方法采用标准裂解法提取K562细胞总蛋白,后续进行丙酮沉淀和三氯乙酸/丙酮沉淀,分别比较一步裂解法所得蛋白和丙酮沉淀法、三氯乙酸/丙酮沉淀法所得蛋白的双向电泳图谱,优选最佳的样品制备方法。此外在等电聚焦时,聚焦电压在传统聚焦条件上进行优化。结果 丙酮沉淀法不仅能减少蛋白降解,而且增加蛋白的溶解性,因此丙酮沉淀法所得蛋白的双向电泳图谱显示出更好的溶解性和重复性,在聚焦条件上对传统聚焦条件的优化使横向和纵向拖尾有明显改善,所得图谱蛋白点更清晰,分离更完全,能获得分辨率较高的双向电泳图谱。结论 在标准裂解液提取蛋白基础上结合丙酮沉淀法更适于制备K562细胞的双向电泳样品,等电聚焦(IEF)电泳时适当延长除盐时间能更有效的分离K562细胞全蛋白,而且后续实验证明此方法同样成功建立了墓头回作用白血病K562细胞蛋白和肿瘤组织蛋白2-DE图谱。Objective To establish the two-dimensional electrophoresis（2-DE） map of K562 cell protein and to further understand the new mechanism of diversifolious patrinia root worked on leukemia cell.Methods Standard lysis buffer was used for extracting total protein,and then precipitated by acetone precipitation and TCA/Acetone precipitation respectively.Besides,the focus voltage was optimized under the traditional 2-DE focus conditions.Results Acetone precipitation not only decreased protein decomposition,but also increased protein solubility,therefore obtaining the 2-DE map with higher resolution and better reproducibility.The optimized focusing condition improved horizontal and vertcal tail obviously,and a high-resolution 2-DE map with clearer protein spots and more complete separation was obtained.Conclusion Acetone precipitation on the basic of standard lysis buffer is more suitable for preparing the 2-DE K562 cell sample.In addition,the longer desalinization time in isoelectric focusing（IEF） can separate whole protein effectively and the optimized methods can be used widely for extraction the protein of tumor tissues.

关 键 词：K562细胞 双向电泳 三氯乙酸/丙酮沉淀 丙酮沉淀 墓头回 

分 类 号：R543.5[医药卫生—心血管疾病]