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急性淋巴细胞白血病白细胞磷酸化蛋白质分析方法的建立*

段春燕，皮 爽1，何 涛，景 莉2，郑小莉

（泸州医学院生物化学教研室/医学分子生物学实验室,四川泸州 646000；1南京大学物理系05级；2泸州医学院附属医院血液内科）

摘 要 目的：建立一种对急性淋巴细胞白血病白细胞磷酸化蛋白质分析方法。方法：分离人新鲜外周血白细胞，并随机分为刺激组和对照组。裂解细胞，用Bradford法测定细胞蛋白质含量，利用亲和层析富集白细胞磷酸化蛋白质，SDS-PAGE分离磷酸化蛋白质，采用Scion Image软件分析电泳结果。结果：①电泳图谱显示每条泳道中蛋白质条带清晰，其相对分子量主要介于26～36kDa以及47～65kDa之间。②EGF刺激后出现有磷酸化蛋白质条带的增减或某些磷酸化蛋白质条带灰度的增强或减弱。结论：采用本研究建立的分析磷酸化蛋白质的方法对急性淋巴细胞白血病白细胞ERK相关磷酸化蛋白质进行动态的分析，其方法较简便、结果可信，可为细胞信息传递磷酸化蛋白质组的研究奠定了基础。

关键词 急性淋巴细胞白血病；SDS-聚丙烯酰胺凝胶电泳；磷酸化蛋白质

*四川省教育厅重点项目（2003A051）

ESTABLISHMENT OF ANALYTIC METHOD FOR PHOSPHOPROTEINS IN LEUKOCYTES FROM ACUTE LYMPHOCYTE LEUKEMIA

Duan Chunyan，et al

Department of Biochemistry,Luzhou Medical College

Abstract Objective: To establish a method to analyze the phosphoproteins in leukocytes from acute lymphocyte leukemia.Methods: Peripheral blood leukocytes were separated from healthy volunteers and patients with ALL . Leukocyte proteins were collected and their concentrations were determined with bradford method. Phosphoproteins in the collected leukocyte proteins were enriched by BDTM phosphoprotein enrichment spin columns. Finally the enriched phosphoproteins were separated by SDS-PAGE, and the electrophoresis maps of SDS-PAGE were analyzed by scion image tool. Results: The SDS-PAGE map of phosphoproteins showed that the pattern of ERK-related phosphoproteins changed obviously in leucocytes of patients with ALL as compared with healthy volunteers. Conclusion: This method is simple and creditable in phosphoprotein analysis for leukocytes of patients with ALL．And it is valuable in functional proteomic analysis for protein phosphorylation modification during cellular signal transduction．

Key words Acute Lymphocyte Leukemia；SDS-PAGE；Phosphoprotein