【摘要】 目的尝试利用质谱技术对肝癌细胞表面自然呈递的MAGE3表位进行检测和分析。方法肝癌细胞和肝细胞取自同1名男性肝癌患者,弱酸洗涤法分离肝癌细胞和癌旁无瘤肝细胞表面所有肽段;利用表位预测法挑选出HLAA2限制的MAGE3理论侯选肽;检测MAGE3mRNA的表达;肽混合物进行高压液相纯化分离和差异比较;筛选出含有肿瘤特异肽的组分进行串联质谱检定和定量分析;最后检测患者体内是否有针对该表位的T细胞反应。结果MAGE3在肝癌细胞样品中表达阳性,在肝细胞中阴性;随后从肝癌细胞样品中检测出自然呈递的MAGE3271279表位抗原肽FLWGPRALV(3839拷贝/细胞),并从患者外周血中检测出针对该表位的特异性T细胞反应。结论本实验从肿瘤组织中检测出自然呈递的MAGE3271279表位,并揭示了该表位的潜在应用价值,同时也初步展示了质谱在肿瘤相关表位检测中快捷、准确的特点,而这些特点对于肿瘤相关表位的鉴定和肽疫苗的设计是非常重要的。更多还原

【Abstract】 Objective To assay the naturally processed MAGE-3 epitopes in tissues of hepatocellular carcinoma (HCC) by Mass Spectrometry.Methods Peptides were isolated by mild acid elution from the surfaces of HCC cells and non-neoplastic liver cells,which were got from a male patient with HCC.Prediction of potential HLA-A2 ligands was carried out from protein sequence of MAGE-3.The mRNA expression of MAGE-3 was detected by RT-PCR.The mixtures of peptides were fractionated by High performance liquid chromatography (HPLC) and a differential comparison was made between the two groups.The fractions containing tumor-specific peptides were analyzed by tandem mass spectrometer (MS).Finally,T cell responses against identified epitope were tested in the patient.Results MAGE-3 transcript was positive in HCC samples and negative in non-neoplastic liver cells.Then a naturally processed MAGE-3_ 271-279 peptide FLWGPRALV (38-39 molecules/cell) was detected in HCC,and specific CD8+ T cell responses to this epitope were also found.Conclusion To our knowledge,this is the first time that the naturally processed MAGE-3_ 271-279 can be isolated and identified from the tumor tissue of HCC patient.Our results implied that MAGE-3_ 271-279 may have some potential value in medicine.This study also revealed the MS-based approach can determine naturally processed peptides in association with tumor rapidly and precisely,which is a prerequisite for efficient epitope identification and vaccine designation.更多还原