摘要
目的探讨来源于人甲胎蛋白（AFP）的细胞毒性T淋巴细胞（CTL）表位多抗原肽（MAP）疫苗对原发性肝癌的抗肿瘤活性。方法体外分离培养人类白细胞抗原（HLA）-A2．1阳性的健康志愿者外周血和HIA．A2．1转基因小鼠骨髓来源的成熟树突细胞（DC），用DC负载多肽疫苗，体内外诱导效应细胞，采用标准4h51Cr释放实验和酶联免疫斑点技术（ELISPOT）检测CTL活性。结果（1）在最高效靶比时CTL表位MAP疫苗诱导的效应细胞对AFP阳性并且HLA-A2．1阳性的Hep3B肝癌细胞的杀伤率显著高于单肽表位诱导的效应细胞（73．5％±7．9％比45．6％±6．9％，P〈0．01）。（2）最高效靶比时AFP表位MAP疫苗和单肽疫苗诱导的效应细胞对AFP阴性而HLA．A2．1阳性的PLC／PRF／5肝癌细胞的杀伤率与阴性肽对照组比较，差异均无统计学意义（9．3％±3．9％、8．1％±2．8％比8．3％±2．6％，均P〉0．05）；MAP组和对应单肽组效应细胞对转染AFP全长cDNA的PLC／PRF／5／Ad—AFP肝癌细胞的杀伤率均显著高于阴性肽对照组（74．8％±10．5％、51．4％±12．6％比4．2％±1．3％，均P〈0．01），而且MAP诱导的CTL对PLC／PRF／5／AFP肝癌细胞的杀伤率显著高于相应单肽（P〈0．01）。（3）最高效靶比时AFP表位MAP疫苗和单肽疫苗诱导的效应细胞对AFP阳性但HLA—A2．1阴性的HepG2肝癌细胞的杀伤率与阴性肽对照组差异均无统计学意义（均P〉0．05）；其对转染了HLA．A2．1全长cDNA的HepG2／HLA．A2．1细胞的杀伤率则均显著高于阴性肽对照组（71．8％±8．6％、46．5％±6．5％比4．1％±1．1％，均P〈0．01），MAP诱导的CTL对HepG2／HLA—A2．1的杀伤率显著高于相应单肽（P〈0．01）。（4）AFP表位MAP疫苗和单肽疫苗诱导的效应细胞产生的IFN-γ斑点数均显著高于阴性对照肽[（158±23）、（78±12）比（3±1）斑点／10’个细胞，均P〈0．01]，MAP的斑点数与阳性对照组[（166±32）斑点／10’个细胞]差异无统计学意义（P〉0．05），MAP的斑点数亦显著高于其单肽[（78±12）斑点／10^5个细胞，P〈0．01]。结论AFPMAP诱导的CTL反应强于相应单肽诱导的CTL反应，对AFP阳性且HLA—A2．1相匹配的肿瘤细胞具有明显的杀伤效应；对AFP阴性的肿瘤细胞不具有杀伤效应。
Objective To explore the antitumor effects of multiple antigen peptide （MAP） vaccine from ot-fetoprotein （AFP） through AFP-speeifie cytotoxic T lymphocyte （CTL） against hepatoma in vitro and ex vivo. Methods Dendritic cells （DC） were generated from human perpheral blood mononuelear cells （PBMC） and HLA-A2. 1-transgenie murine bone marrow. The AFP-specific CTL were induced by MAP loaded DC and the corresponding linear peptides from human AFP. The lysis rate of effectors to hepatoma cells were tested by 4 h 51Cr release assay. And enzyme-linked immunosorbent spot（ELISPOT） was used to test the interferon （IFN-γ, release of effector cells. Results The specific lysis rate of effectors induced by AFP epitopie MAP vaccines to Hep3B cells （AFP＋ , HLA-A2, 1＋） at the highest effeetor/target （E/T） ratio was significantly higher than linear peptide vaccine （73.5% ±7.9% vs 45.6% ±6.9% , P 〈0. 01 ） The effeetors induced by AFP epitopic MAP vaccine and linear peptide vaccine could not lyze the AFP- negative PLC/PRF/5 liver cancer cells versus the negative control group at the highest E/T （ 9. 3% ±3.9% ,8. 1% ±2. 8% vs 8.3% ±2. 6%, both P 〉0.05）. But the effectors induced by AFP epitopic MAP vaccine and linear peptide vaccine could lyse PLC/PRF/5 liver cancer ceils transfected with eDNA of AFP versus the negative control group （74. 8% ± 10. 5%, 51.4% ± 12. 6% vs 4. 2% ± 1.3%, both P 〈0. 01 ）. And the specific lysis rate of effectors induced by AFP epitopie MAP vaccines was significantly higher than the corresponding linear peptide vaccine （ P 〈 0. 01 ） . Compared with the negative control group, the effeetors could not lyse HepG2 liver cancer cells, a HLA-A2. 1 negative cell line （ both P 〉 0. 05 ）. But the effeetors could lyse HepG2 cells transfected with eDNA of HLA-A2. 1 （71.8% ±8. 6%, 46. 5% ±6. 5% vs 4. 1% ± 1.1%, both P 〈 0. 01 ）. And the specific lysis rate of effeetors induced by MAP vaccine was significantly higher than the corresponding linear peptide vaccine （P 〈0. 01 ）. ELISPOT test showed that the capability of enhancing IFN-γ release of human AFP MAPs was stronger than that of the AFP linear peptides. The spots count of MAP vaccine group （（ 158 ± 23 ） spots/10s cells） or linear peptide vaccine group （（78 ± 12 ） spots/105 eel/s） were significantly higher than the negative control group （（3 ±1 ） spots/10s cells） （all P 〈 0. 01 ）. The spots count of the positive control group （ （ 166 ± 32 ） spots/105 cells） showed no significant difference with the AFP MAP vaccine group （ P 〉 0. 05 ）. And the spots count of MAP vaccine group were significantly higher than the corresponding linear peptide vaccine group （（78 ± 12 ） spots/10s cells, P 〈 0. 01 ）. Conclusions AFP multiple antigen peptides elicit not only more powerful specific anti-tumor immune responses but also stronger non-specific anti-tumor immune activities than their corresponding linear peptides. These findings will provide theoretical rationales for their clinical applications.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2012年第16期1138-1142,共5页
National Medical Journal of China
关键词
甲胎蛋白类
肝肿瘤
疫苗
免疫效应
alpha-Fetoproteins
Liver neoplasms
Vaccines
Immunological effect
