CAJ | 학술논문

目的：肿瘤抗原的存在是机体识别肿瘤并激活免疫系统的物质基础，机体抗肿瘤免疫以细胞免疫为主，故寻找被T细胞识别的肝癌细胞抗原肽，为肝癌免疫治疗奠定基础。方法：对于肝癌细胞系HHCC(HLA-A2,A29,B7,B51)，应用细胞膜酸洗技术使抗原肽从细胞膜表面脱落，经过凝胶层析，反相高效液相色谱层析得到不同组份多肽，高效液相色谱-质谱仪联用技术获得抗原肽的一级结构，通过氨基酸锚定位点分析，预测其HLA配体类型；互联网上氨基酸同源性分析确定其同源性序列；人工合成抗原肽，HLA同型树突状细胞递呈合成抗原肽刺激特异性CTL反应，51Cr杀伤实验检测CTL对肿瘤细胞系的杀伤作用。结果：经过反相高效液相色谱层析后可以获得10多个组份，其中一个组份经过液质联用鉴定和氨基酸同源性分析确定其序列为EPVTKAEML，是黑色素瘤抗原-1，MAGE-1(121-129)表位，由HLA-B7分子递呈，体外诱导实验表明其可以诱导出较强的CTL反应。结论：液质联用技术是寻找抗原肽的有效方法，MAGE-1抗原肽EPVTKAEML对于HLA-B7阳性及MAGE-1阳性的肝癌患者具有潜在的免疫治疗作用。
목적：종류항원적존재시궤체식별종류병격활면역계통적물질기출，궤체항종류면역이세포면역위주，고심조피T세포식별적간암세포항원태，위간암면역치료전정기출。방법：대우간암세포계HHCC(HLA-A2,A29,B7,B51)，응용세포막산세기술사항원태종세포막표면탈락，경과응효층석，반상고효액상색보층석득도불동조빈다태，고효액상색보-질보의련용기술획득항원태적일급결구，통과안기산묘정위점분석，예측기HLA배체류형；호련망상안기산동원성분석학정기동원성서렬；인공합성항원태，HLA동형수돌상세포체정합성항원태자격특이성CTL반응，51Cr살상실험검측CTL대종류세포계적살상작용。결과：경과반상고효액상색보층석후가이획득10다개조빈，기중일개조빈경과액질련용감정화안기산동원성분석학정기서렬위EPVTKAEML，시흑색소류항원-1，MAGE-1(121-129)표위，유HLA-B7분자체정，체외유도실험표명기가이유도출교강적CTL반응。결론：액질련용기술시심조항원태적유효방법，MAGE-1항원태EPVTKAEML대우HLA-B7양성급MAGE-1양성적간암환자구유잠재적면역치료작용。
Objective: The current approach to immu motherapy is mainly reliant on the role of T lymphocyte and tumor antigen recognized by T lymphocyte. To broaden the clinical applicability of peptide-based immunotherapy in human hepatocellular carcinomas, there is a need to isolate and identify tumor antigen peptides bound to HLA class I molecules on the human hepatocellular carcinomas. Methods: The prime structure characterization of HLA binding peptide by a human hepatocellular carcinoma cell line with a HLA-A2,A29,B7,B51 was performed. Extract of MHC-associated peptides by mild acid wash of viable hepatocellular carcinomas cells, and collected by gel filtration, then, fractionated by reversed phase high pressured liquid chromatography (RP-HPLC). HPLC-fractionated samples were sequenced and identified by HPLC-MS-MS (tandom mass spectrometry ). Protein database in the intemet were used as additional tools for structure analysis and for determination of the protein source of the eluted peptides,potential peptide binding HLA alleles had been identified using a computer program by comparing anchor amino acid residues. Allologous dendritic cells (DCs) having the same HLA alleles pulsed the synthetic peptide, PBMCs were stimulated by peptide-pulsed autologous DCs. The specific cytotoxicity against the hepatocellular carcinoma cell line were determined by 51Cr release assay.Results: RP-HPLC showed that there were teens different fractions of peptides derived from HHCC human hepatocellular carcinoma cell line,HPLC-ESI-MS-MS showed the amino acid sequence of the fractions. One of the most promising candidates for T cell epitope is nonamers peptide EPVTKAMEL, derived from MAGEl121-129, had the HLA-B7 peptide binding motify. PBMC cocultured with the peptide-pulsed DCs could induce the relevant peptide specific CTLs,moreover, they showed specific cytotoxicity against HHCC cell line. Conclusion: Sensitive sequencing by HPLC-MS may provide a powerful method of identifying tumor specific antigenic peptides, nonamers peptide EPVTKAEML, can be used for hepatocellular carcinoma vaccine strategies with confidence that it is identical to the naturally processed peptide epitopes presented at the surface of hepatoma cells in association with HLA-B7 molecules.