摘要目的 分析健康对照组(Control组)与病毒性心肌炎组(VMC组)血浆外泌体中的差异蛋白质组,筛选可能与VMC早期诊断密切相关的蛋白标志物.方法 分别收集河南省人民医院(2016年1月至2017年12月)Control组健康人及VMC组患者血浆标本各50份,按照随机数字表法选取Control组(n=5)与VMC组(n=5)空腹血浆标本各1 ml,分别利用超速离心法提取血浆外泌体后,采用差异凝胶双向电泳(DIGE)技术进行蛋白质分离,经差异分析软件处理后,采集VMC组和Control组之间差异＞2倍的蛋白质点,通过应用基质辅助激光解吸电离-飞行时间/飞行时间质谱(MALDI-TOF/TOF MS)对差异蛋白点进行鉴定.各组随机采取40份血浆外泌体标本,采用VMC特异性差异蛋白进行酶联免疫吸附实验(ELISA)验证.结果 VMC组与Control组血浆外泌体蛋白组相比较,共筛选出10个差异蛋白点,包括8个上调蛋白点,2个下调蛋白点.经质谱鉴定后,表达上调的蛋白包括角蛋白2(KRT2)、角蛋白5(KRT5)、角蛋白9(KRT9)、角蛋白77(KRT77)、角蛋白78(KRT78)、锌-α2糖蛋白(AZGP1)、触珠蛋白(HP)和视黄醇结合蛋白4(RBP4);表达下调的蛋白是CD5抗原样蛋白(CD5L)和补体C1q亚基B(C1QB).选取RBP4进行ELISA验证,结果表明,与Control组[(64.5±2.6)μg/L]相比较,VMC组[(148.7±3.9) μg/L]血浆外泌体中RBP4特异性表达上调(P＜0.05),与DIGE结果一致.结论 成功筛选到10个VMC相关差异蛋白质,RBP4有可能成为VMC早期筛查及诊断的特异性生物标志物.

abstractsObjective To compare and analyze the differentially expressed plasma exosomic proteome between healthy control group (Control group) and viral myocarditis group (VMC group) to search for biomarkers that maybe used for early diagnosis of VMC.Methods Fifty plasma samples of Control group and VMC group were collected respectively from Henan Provincial People's Hospital (from January 2016 to December 2017),and then 5 samples (1 ml) of each group were selected randomly,after exosomes extraction with ultra-centrifugation,difference gel electrophoresis (DIGE) was used to isolate the total proteins,and then the protein spots with more than 2-fold changes between VMC and Control group were picked up after the software analysis,afterward,the varied proteins were identified by MALDI-TOF/TOF mass spectrometry.Finally,the specifically related protein was selected to be verified by ELISA with the plasma exosomic samples of Control (n=40) and VMC (n=40).Results A total of 10 varied protein spots were found including 8 up-regulated proteins and 2 down-regulated proteins between VMC and Control group.After MS analysis,the up-regulated proteins in VMC group contained KRT2,KRT5,KRT9,KRT77,KRT78,AZGP1,HP and RBP4,whereas the down-regulated ones were CD5L and C1QB.RBP4 was selected to validate by ELISA analysis,and the corresponding results showed that RBP4 was increased specifically in plasma exosomes of VMC group (P＜0.05) after comparing with Control group,which was consistent with DIGE.Conclusion Ten proteins related to VMC are detected in total,and RBP4 might serve as a potential specific biomarker for early screening and diagnosis of VMC.