儿童肝母细胞瘤潜在标志物载脂蛋白A-I的筛选及鉴定

Screening and identification of apolipoprotein A-I as a potential marker for hepatoblastoma in children

摘要:

目的 筛选并鉴定出小儿肝母细胞瘤（HB）血清生物学标记物，以助HB的早期诊断。方法 使用弱阳离子磁珠技术处理30例HB患儿、20例全身炎症反应综合征（SIRS）患儿和20例正常儿童的血清样品，利用表面增强激光解吸电离飞行时间质谱（SELDI-TOF-MS）平台筛选出肿瘤组和正常组的差异性蛋白。SDS-PAGE纯化分离出目标蛋白质后，应用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）鉴定出目标蛋白质的氨基酸序列，并通过SwissProt数据库查找与之匹配的蛋白质。最后使用Real-time PCR和ELISA技术验证该蛋白质的表达情况。结果 SELDI-TOF-MS筛选并排除炎症因子干扰后，发现HB组和正常组存在质合比为9 348 Da的差异性蛋白质，在HB组的表达较低（P < 0.05）。经鉴定此蛋白为载脂蛋白A-I（Apo A-I）。Real-time PCR和ELISA验证Apo A-I基因和蛋白在HB组均低表达，在正常组高表达。结论 Apo A-I可作为HB非炎症性蛋白质标记物，对其早期诊断有意义。

Abstract:

Objective To screen and identify serum biomarkers for childhood hepatoblastoma (HB). Methods The serum samples from 30 children with hepatoblastoma (HB), 20 children with systemic inflammatory response syndrome, and 20 normal children were treated with magnetic bead-based weak cation exchange chromatography. The platform of surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) was used to eliminate the interference of inflammatory factors and to screen out the differentially expressed proteins in serum between tumor group and normal group. After the purification and separation of target proteins were performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry was used to determine their amino acid sequences. The SwissProt database was searched for matched proteins. Finally, real-time PCR and ELISA were used to verify and measure the expression of target proteins. Results After SELDI-TOF-MS was used for screening and elimination of the interference of inflammatory factors, a differentially expression protein with a mass-to-charge ratio of 9 348 Da was found in serum between HB group and normal group, and the HB group had significantly lower expression of this protein than the normal group (P < 0.05). This protein was identified as apolipoprotein A-1 (Apo A-I). Real-time PCR and ELISA verified the low mRNA and protein expression of Apo A-I in serum in the HB group and high expression in serum in the normal group. Conclusions Apo A-I can be used as a non-inflammatory protein marker for HB and has a certain value in the early diagnosis of HB.