利用DIGE和RP-HPLC-ESI/MS技术寻找慢性移植肾失功患者血清标志物  

Finding the serum biomarkers of chronic renal allograft dysfunction by DIGE and RP-HPLC-ESI/MS techniques

作　　者：高英[1] 吴轲[1] 徐逸[1] 周鸿敏[1] 何文涛[1] 张维娜[1] 林星光[1] 蔡兰军[1] 雒真龙[1] 方泽民[1] 郭晖[1] 周绍棠[1] 昌盛[1] 杜敦峰[1] 陈忠华 

机构地区：[1]华中科技大学同济医学院附属同济医院器官移植研究所,教育部,卫生部器官移植重点实验室,武汉市430030

出　　处：《实用医学杂志》2009年第20期3363-3366,共4页The Journal of Practical Medicine

基　　金："973"计划专项基金(编号:2009CB522407)

摘　　要：目的:利用蛋白组学二维差异性凝胶电泳(2-DDIGE)和反相高效液相色谱法(RP-HPLC)联合电喷射离子化质谱(ESI/MS)技术寻找慢性移植肾失功患者血清生物标志物。方法:血清样本分成4组,慢性移植物失功组(CGD组)、移植后长期肾功能稳定组(SRF组)、急性排斥组(AR组)和健康对照组(N组)。血清样本经多重亲和排除柱去除高丰度蛋白处理后,使用2-DDIGE技术找到差异蛋白点。切下这些差异蛋白点,胰蛋白酶消化,经RP-HPLC-ESI/MS分析鉴定,在另一独立样本中对鉴定出的差异蛋白Galectin-7进行ELISA验证。结果:在CGD组患者血清中差异表达的蛋白共有39个,质谱鉴定出22个,包括载脂蛋白A-I前体,补体C4-A前体,Galectin-7等。ELISA结果提示CGD组患者血清中Galectin-7的表达较SRF组和N组有明显增高。结论:找到的这些差异蛋白具有不同的生物功能,为大样本、多中心的验证奠定了基础。Galectin-7可能是CGD的血清标志物,并对CGD的发病机制和治疗策略提供新的思路。Objective To identify the serum biomarkers of chronic renal allograft dysfunction （CGD） by twodimensional differential in-gel electrophoresis （2-D DIGE） and reversed phase high-performance liquid chromatography （RP-HPLC） followed by electrospray ionization mass spectrometry （ESI-MS）. Methods Serum samples, which were divided into 4 groups： CGD group, long-term stable renal function group （SRF）, acute rejection group （AR）, and normal volunteer group （N）, were firstly processed by multiple affinity removal column to selectively remove the highest abundance proteins. Then the differential protein spots, which were found by 2-D DIGE, were excised and then digested by tupsin, and then analyzed by RP-HPLC-ESI/MS. Finally, Galectin-7, one of these identified proteins, was confirmed by ELISA analysis in an independent set of serum samples. Results A total of 39 protein spots were found to be differentially expressed in samples in CGD group, in which 22 proteins, including apolipoprotcin A-I precursor, complement C4-A precursor, Galectin-7, etc, were identified. ELISA analysis result showed that Galetin-7 was significantly up-regulated in samples in CGD group as compared to those in SRF group and those in N group. Conclusion The differentially expressed proteins are invoh,ed in different physiologic functions. Galectin-7 may be serum biomarkers for CGD, which may provide a new insight into the pathogenesis and treating strategy of CGD.

关 键 词：肾移植 慢性移植物失功 蛋白组学 血清 

分 类 号：R699[医药卫生—泌尿科学]