稳定型心绞痛患者血浆差异蛋白的筛选及验证

Screening and verification of plasma biomarkers for stable angina pectoris:a differential proteomic analysis

目的分析稳定型心绞痛(SAP)患者与健康对照组血浆中差异表达的蛋白质组,筛选可能与SAP诊断密切相关的血浆生物标记物。方法采集南方医科大学第三附属医院SAP血浆标本60例,另收集体检科收集的血浆标本作为正常对照组(n=60)。下面不变,做凝胶电泳是将各组的血混合的做实验的。取SAP患者组与健康对照组空腹血浆标本各20例(各100 mL),分别等量混合成2组样本,去除血浆高丰度蛋白后,利用双向凝胶电泳技术进行蛋白分离,经差异软件分析后,挖取变化2倍以上的差异蛋白质点,利用基质辅助激光解析电离-飞行时间/飞行时间质谱对差异蛋白点进行鉴定。每组随机各取40例样本,选取差异蛋白血红蛋白δ亚基进行酶联免疫吸附实验验证。结果 SAP患者与健康人血浆相比较,筛选出表达量差异2倍以上的差异蛋白共7种,表达上调的蛋白包括血清白蛋白、血红蛋白α亚基和血红蛋白δ亚基;表达下调的蛋白包括载脂蛋白L1、载脂蛋白C3、载脂蛋白E和补体C4-B。ELISA验证结果表明血红蛋白δ亚基在SAP组样本中表达上调,与双向凝胶电泳结果一致。结论血浆血红蛋白δ亚基有可能成为稳定型心绞痛早期筛查及诊断的特异性生物标记物。

Objective To compare and analyze the differentially expressed plasma proteome between patients with stable angina pectoris（SAP） and healthy donors to identify the biomarkers for early diagnosis of SAP.Methods Plasma samples from60 patients with SAP and 60 healthy controls were collected.Twenty samples（100 mL each） randomly selected from each group were pooled and after removing high-abundance proteins from the pooled plasma,two-dimensional gel electrophoresis（2DE） was performed to isolate the total proteins.The protein spots with more than 2-fold changes were selected after 2D analysis using software,and the differentially expressed proteins were identified by MALDI-TOF/TOF mass spectrometer.ELISA was performed to detect hemoglobin subunit delta（HBD） levels in 40 randomly selected samples from each group for verification of the results of 2DE.Results A total of 7 differentially expressed proteins were found in plasma samples from patients with SAP,including 3 up-regulated proteins（serum albumin,hemoglobin subunit alpha and hemoglobin subunit delta,） and 4 down-regulated ones（apolipoprotein L1,apolipoprotein C3,apolipoprotein E and complement C4-B）.ELISA results showed that HBD level was increased in SAP plasma,which was consistent with the results of 2 DE.Conclusion Patients with SAP have different plasma protein profiles from those of healthy controls,and HBD may serve as a potential specific biomarker for early diagnosis of SAP.