人类基因组编码蛋白高通量芯片筛选原发性胆汁性肝硬化血清标志物

摘要

Objective To screen serum markers in patients with PBC by high-throughput protein chips encoded by the human genome. Methods High-throughput protein chips (contains a total of 38 400protein spots, including 17 718 human genes encoding proteins) were used to screen sera from 21 PBC patients, 20 disease control patients and 10 normal controls. Bioinformatics software was used to analyze information and statistical software was used to analyze the data to confirm the serum markers of PBC. Results The detection rate of protein spots using anti-GST antibody on the chip was 97. 6%, and the signal intensity correlation coefficient of double protein spots was 0. 98. Four serum markers( PDHA1, DBT,DLAT and HK1 )were screened by high-throughput protein chips between PBC group and the control group with a statistically significant. The positive rate of the four markers in the three groups was 66. 67% ( 14/21), 5.00% (1/20) and 0(0/10); 57. 14% ( 12/21 ), 5.00% (1/20) and 0(0/10); 52. 38% ( 11/21 ),0(0/20) and 0(0/10); 52. 38% ( 11/21 ), 0(0/20) and 0(0/10) respectively. All the four markers were different in the three groups with statistically significant (PDHA1 :x2 = 16. 79, P ＜0. 01 ;Fisher exact test,P=0. 000; DBT:x2 =12.86, P＜0. 01;Fisher exact test, P=0. 004; DLAT and HK1:Fisher exact test,P ＜0. 01 or 0. 05). Of those markers, antibodies to PDHA1, DBT and DLAT were the component of AMAM2 which had been used as the marker of PBC. Antibody to HK1 was identified as new marker of PBC,whose sensitivity to PBC was 52. 38% and specificity was 100. 00%. There were no serum marker were screened between the AMA-M2 positive and negative PBC patients. Only antibody to CENPB was identified to be significantly expressed between the ACA positive and negative PBC patients ( Fisher exact test, P =0. 000). Conclusions High-throughput protein chip encoded by the human gene is a technology for quick and comprehensive screening of new markers of PBC. Antibodies to HK1 could be used as new marker for PBC with highly sensitivity and specificity. No serum marker is found between the AMA-M2 positive and negative PBC patients whereas only antibody to CENPB is identified as marker between the ACA positive and negative PBC patients.%目的 探讨用人类基因组编码蛋白高通量芯片(以下简称"高通量蛋白芯片")筛选出有诊断价值的PBC血清标志物的应用价值.方法 用高通量蛋白芯片(包含17 718个人类基因编码蛋白,共有38 400个蛋白点)筛选21例PBC患者、20例疾病对照患者(AIH 7例,病毒性肝炎8例,其他自身免疫性疾病5例)和10名健康对照者血清,并用生物信息学软件提取筛选信息后,经统计软件分析确定有价值的诊断PBC的血清标志物.结果 用抗GST抗体对高通量蛋白芯片进行检测的结果显示,该芯片蛋白点的检出率为97.6%,蛋白复点间检测信号强度的相关系数为0.98.用高通量蛋白芯片从PBC组、疾病对照组和健康对照组中筛选出4个差异有统计学意义的标志物(PDHA1、DBT、DLAT和HK1),他们在3组中的阳性率分别为66.67%(14/21)、5.00%(1/20)和0(0/10),57.14%(12/21)、5.00%(1/20)和0(0/10),52.38%(11/21)、0(0/20)和0(0/10),52.38%(11/21)、0(0/20)和0(0/10);3组4项指标分别比较的差异均有统计学意义(PDHA1:x2=16.79,P＜0.01;Fisher精确检验,P=0.000;DBT:x2=12.86,P＜0.01;Fisher精确检验,P=0.004;DLAT和HK1:Fisher精确检验,P均分别为0.01、0.05).其中,针对PDHA1、DBT和DLAT蛋白的抗体为现已使用的PBC标志物-AMA-M2组成部分;针对HK1蛋白的抗体为新发现的PBC标志物,其对PBC的诊断敏感度为52.38%,特异度为100.00%.AMA-M2阳性与AMA-M2阴性PBC患者中未发现差异有统计学意义的血清标志物.经Fisher精确检验,ACA阳性与ACA阴性PBC患者间仅针对着丝粒B(CENPB)蛋白的抗体差异有统计学意义(Fisher精确检验,P=0.000).结论 高通量蛋白芯片是一种快速全面筛选诊断PBC标志物的技术.针对HK1蛋白的抗体对PBC具有较好的敏感度和特异度,可作为PBC新标志物.AMA-M2阳性与AMA-M2阴性PBC患者间未发现具有统计学意义的血清标志物,而针对CENPB蛋白的抗体可作为ACA阳性与ACA阴性PBC患者筛选的标志物.