HLA-A2限制性Survivin点突变肽诱导特异性抗肝癌CTL反应  

HLA-A2-restricted point-mutated Survivin peptide elicited specific cytotoxic T lymphocyte reaction against hepatocarcinoma

作　　者：沈晗[1,2] 黄昭亮[1] 陈晓华 林燕梅[1] 王腾[1] 邵红伟[1] 黄树林[1] 

机构地区：[1]广东药学院生命科学与生物制药学院,广州510006 [2]南方医科大学,510515 [3]广东省广州市番禺区中心医院肿瘤血液科

出　　处：《中华微生物学和免疫学杂志》2012年第8期696-701,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家科技重大专项资助项目（2009ZX09103-708）;国家自然科学基金资助项目（31100664）;广东省自然科学基金资助项目（10151022401000024）

摘　　要：目的探讨HLA—A2限制性Survivin点突变抗原肽体外诱导CTLs的抗肝癌作用。方法生物信息学软件BIMAS和SYFPEITHI用来鉴定点突变的HLA-A2限制性Survivin抗原九肽；流式细胞术检测突变肽与HLA-A2分子结合效率；肽体外刺激肝癌患者腹水来源的肿瘤浸润淋巴细胞（TILs）诱导反应性CTLs，用流式细胞术及ELISA检测反应性CTLs释放IFN-γ情况；肽诱导的CTLs与肝癌细胞系HepG2及BEL-7402共孵育，CytoTox96@非放射性细胞毒性检测法检测对肿瘤细胞的裂解情况，倒置相差显微镜观察肝癌细胞形态学变化。结果生物信息学分析筛选出与MHC分子结合效率评分显著提高的点突变Survivin抗原九肽Sur79M2（KMSSGCAFL），流式细胞术检测证实负载Sur79M2的T2细胞，HLA-A2表达率显著提高，经Sur79M2体外刺激诱导的CTLs与靶细胞共孵育后能释放较高水平的IFN-γ，Sur79M2诱导的CTLs能通过HIA．A2限制性机制有效杀伤肝癌细胞系HepG2，对HLA—A2阴性的BEL-7402细胞无明显杀伤作用。结论点突变肽Sur79M2能在体外诱导反应性CTLs产生，该CTLs能以HLA-A2限制性方式有效杀伤肝癌细胞系。Objective To investigate the anti/hepatocarcinoma（anti-HCC） function of HLA-A2- restricted point-mutated Survivin peptide induced CTLs. Methods The HLA-A2-restricted Survivin non- apeptides were evaluated using bioinformatics software. The binding affinity of Survivin peptide to HLA-A2 molecular was determined with flow cytometry analysis. After peptide-induced CTLs were generated in vitro, flow cytometry and ELISA were performed to detect the levels of IFN-T, which were secreted by reactive CTLs. Peptide-induced CTLs were co-cultured with hepatoma cell lines HepG2 and BEL-7402. The rates of tumor cells lysis were assayed using CytoTox 96 and the morphological changes of tumor cells were observed with inverted phase contrast microscope. Results Point-mutated Survivin nonapeptide Sur79M2 （KMSSG- CAFL） was filtered out, which was shown higher scores compared with the wild-type peptide Sur79. Consist- ent with the results of software analysis, Sur79M2 showed higher binding ability in T2 binding assays. At the same time, Sur79M2-induced CTLs couM release a large number of IFN-γ after incubated with target cells rather than Sur79. When co-cultured with HCC cell lines HepG2 and BEL-7402, Sur79M2-induced CTLs ef- fectively lysis HepG2 on HLA-A2-restricted manner without killing effect on BEL-7402 that do not express HLA-A2 molecules. Conclusion Sur79M2 could elicited specific cytotoxic T lymphocytes in vitro, which were able to specifically kill HCC cell lines on HLA-A2-restricted manner.

关 键 词：SURVIVIN 抗肝癌 点突变肽 HLA-A2限制性 

分 类 号：R735.7[医药卫生—肿瘤]