同位素标记相对和绝对定量技术在家族性瘢痕疙瘩蛋白质组学研究中的应用

The application of iTRAQ quantitative proteomics in familial keloid

目的 通过比较家族聚集性瘢痕疙瘩（FK）和散发性瘢痕疙瘩（SK）、增生性瘢痕（HS）、正常皮肤（NS）组织样本间蛋白质组表达的差异,筛选出与FK关系密切的蛋白,以期找到其特异性标志物,探索相关发病机制.方法 应用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation,iTRAQ）技术,对FK、SK、HS、NS 4组,每组6例样本,分别进行酶解、标记及串联质谱分析,进行分子生物学验证及生物信息学分析.并随机挑选2种差异蛋白以蛋白质印迹法进行验证.采用SPSS 16.O软件,用独立样本t检验对4组蛋白检测结果进行统计学分析,P＜0.05为差异有统计学意义,筛选出变化倍数在1.2倍以上的差异蛋白.结果 测定到蛋白质共2 450种,其中各组间差异蛋白数量及情况：与NS相比,FK特异性表达上调的蛋白共250种,下调的蛋白有281种;SK中特异性表达上调的蛋白共281种,下调的蛋白有232种;HS中特异性表达上调的蛋白共199种,下调的蛋白有233种.与SK相比,FK特异性表达上调的蛋白共64种,下调的蛋白有164种;HS特异性表达上调的蛋白共79种,下调的蛋白有169种;与HS相比,FK特异性表达上调的蛋白共124种,下调的蛋白有115种.这些差异蛋白在细胞外基质、细胞黏附及生物代谢等多条重要信号转导通路中集中分布.蛋白印迹法验证结果表明2种差异蛋白（P3H1和RCN-3）变化趋势与iTRAQ技术检测结果相同.结论 iTRAQ技术能较好地显示瘢痕疙瘩、增生性瘢痕与正常皮肤组织间的蛋白质表达差异,将有助于揭示其发病机制,找到其特异性标志物用于诊断和治疗.

Objective To identify the special biomarkers and the differentially expressed proteins in keloid tissue and to explore the pathogenesis characteristics of familial keloid by comparing the protein expression differences among familial keloid（FK）,sporadic keloid （SK）,hypertrophy scar （HS）,normal scar （NS）.Methods The tissue specimens of FK,SK,HS and NS（6 specimens in each group）,were digested,taged and analysed using quantitative proteomic isobaric tags for relative and absolute quantitation （iTRAQ） labeling technology.A difference greater than 1.2 folds and P 〈 0.05 were selection criteria for differencial expression proteins.Then bioinformatics analysis was applied.The expressions of 2 differential proteins were validated using Western Blot analysis.Results A total of 2 450 differentially expressed proteins were identified.Compared with NS,250 up-regulated and 281 down-regulated proteins were identified in FK;281 up-regulated and 232 down-regulated proteins were identified in SK;199 up-regulated and 233 down-regulated proteins were identified in HS.Compared with SK,64 up-regulated and 164 downregulated proteins were identified in FK;79 up-regulated and 169 down-regulated proteins were identified in HS.Compared with HS,124 up-regulated and 115 down-regulated proteins were identified in FK.These different proteins were concentrated in several vital signal pathways such as the extracellular matrix protein,cell adhesion and biological metabolism pathways.The variation trend of 2 differentially expressed proteins （P3H1 and RCN-3） were validated by Western Blot.Conclusions Proteomic analysis can identify the proteins with variance of keloid,hypertrophy scar and normal skin.Further investigations of these differential proteins may reveal the pathogenesis of keloid and provide new treatments for it.