双向凝胶电泳联合MALDI-TOF/TOF MS技术探寻狼疮性肾炎的血清标志物

Exploring Serum Protein Biomarkers of Lupus Nephritis by Using Two-Dimensional Gel Electrophoresis Combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry

目的:通过双向凝胶电泳与基质辅助激光解吸电离串联飞行时间质谱(MALDI-TOF/TOF MS)技术初步探寻狼疮性肾炎(LN)患者潜在的血清分子标志物,为阐明LN的发病机制奠定基础。方法:将40例LN患者分为活动组和缓解组,每组20例;另选择IgA肾病患者和健康志愿者各20例,分别作为IgAN组和健康对照组。采用双向凝胶电泳技术分离血清蛋白质,并采用MALDI-TOF/TOF MS鉴定差异表达的蛋白质。结果:共发现50个差异表达蛋白质。与健康对照组相比,LN活动组和LN缓解组共发现23个差异表达的蛋白质,上调8个、下调15个;与IgAN组相比,LN活动组和LN缓解组共发现18个差异表达的蛋白质,上调13个,下调5个;与LN缓解组相比,LN活动组表达上调和下调的蛋白质分别为4个和5个。在鉴定出的50个差异表达蛋白中,血清淀粉样蛋白A(SAA)在LN活动组中的表达高于其他组,complement component C4A在LN活动组中的表达低于其他组;chain B(solution structure of double super helix model)在LN缓解组的表达高于其他组;与健康对照组相比,vitamin D-binding protein isoform1 precursor、chain A(crystal structure of uncomplexed vitamin D-binding protein)和chain B(a covalent dimer of transthyretin that affects the amyloid Pathway)等在LN活动组和LN缓解组的表达均上调,而vitronectin precursor、ficolin-2isoform a precursor和chain A(crystal structure of the catalytic domain of human complement C1s protease)则相反;与IgAN组相比,lipoprotein CIII和vitronectin precursor在LN活动组和LN缓解组的表达均上调。结论:双向凝胶电泳与MALDI-TOF/TOF MS联用技术有效实现了对LN患者血清差异表达蛋白质的筛选与鉴定。这些差异表达的蛋白质可作为生物标志物用于LN的无创性诊断和评估,对这些蛋白质的进一步研究将有助于更好地了解LN的发病机制。

Objective： To explore the potential serum biomarkers of patients with lupus nephritis（LN） by using twcdimensional electrophoresis combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF/TOF MS）, so as to lay the foundation for illuminating pathogenesis. Methods： A total of 40 LN patients were divided into two groups, the active LN group and the inactive LN group, with 20 in each. In addition, 20 IgA nephritis patients and 20 healthy volunteers were enrolled as IgAN group and healthy control group. Two-dimensional gel eiectrophoresis was used to separate and analyze the serum proteins, and MALDI-TOF/TOF MS was applied to the identification of the differentially expressed pro teins. Results： A total of fifty differentially expressed proteins were identified. Compared with that in healthy control group, 23 differentially expressed proteins were discovered in active LN group and inactive LN group, among which, 8 proteins were up-regulated and 15 proteins were down-regulated. And 18 differentially expressed proteins, compared with IgA nephritis group, were found in active LN group and inactive LN group, including 13 up-regulated proteins and 5 down-regulated proteins. Fur- thermore, the number of upregulated and down-regulated proteins in active LN group, compared with those in inactive LN group, were 4 and 5, respectively. Among the 50 identified differentially expressed proteins, the expression of serum amyloid protein A（ SAA ） in active LN group was higher than that in the other groups while the expression of complement component CAA in active LN group was lower than that in the other groups. And the expression of chain B （solution structure of double super helix model） in the inactive LN group was higher than that in the other groups. Compared with that in healthy control group, the expression of vitamin D-binding protein isoform 1 precursor, chain A（crystal structure of uncomplexed vitamin D- binding protein） and chain B （a covalent dimer of transthyretin that affects the amyloid pathway） was up-regulated in both ac- tive LN group and inactive LN group, while the expression of the vitronectin precursor, ficolin-2 isoform a precursor and chain A （crystal structure of the catalytic domain of human complement Cls protease） was down regulated. Compared with that in IgA nephritis group, the expression of lipoprotein CIII and vitronectin precursor was up-regulated in both active LN group and inactive LN group. Conclusions： Combination of two-dimensional gel electrophoresis and MALDI-TOF/TOF MS is effective for screening and identification of differentially expressed proteins in serum from LN patients. These differentially expressed pro- teins could be used as biomarkers for noninvasive diagnosis and evaluation of LN. Further study on these proteins would be conducive to understanding the pathogenesis of LN.