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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 patients with chronic graft dysplasia by proteomic two-dimensional differential gel electrophoresis (2-DDIGE) and reverse phase high performance liquid chromatography (RP-HPLC) combined with electrospray ionization mass spectrometry Serum biomarker. Methods: Serum samples were divided into four groups: CGD group, SRF group, AR group and N group. Serum samples were subjected to multiple affinity exclusion columns to remove the high-abundance proteins and 2-DDIGE was used to find differential protein spots. These differentially expressed proteins were excised and trypsin digested, identified by RP-HPLC-ESI / MS analysis, and verified in a separate sample of the identified differential protein Galectin-7. Results: There were 39 differentially expressed proteins in serum of CGD patients and 22 of them were identified by mass spectrometry, including apolipoprotein A-I precursor, complement C4-A precursor, Galectin-7 and so on. The results of ELISA indicated that the expression of Galectin-7 in serum of CGD patients was significantly higher than that of SRF and N groups. Conclusion: These differential proteins were found to have different biological functions, which laid the foundation for large sample and multicenter validation. Galectin-7 may be a serum marker of CGD, and provide new ideas for the pathogenesis and treatment strategies of CGD.