甲胎蛋白CTL表位多抗原肽疫苗对原发性肝癌的抗肿瘤免疫效应实验观察
갑태단백CTL표위다항원태역묘대원발성간암적항종류면역효응실험관찰
Epitopic multiple antigen peptide from α-fetoprotein elicit antitumor immune response in vitro and ex vivo
  • 万方数据
    中华医学杂志 중화의학잡지
    National Medical Journal of China
    2012年 16期 1138-1142 ,共5页

    范宗江%张树荣

    범종강%장수영

    甲胎蛋白类%肝肿瘤%疫苗%免疫效应 甲胎蛋白類%肝腫瘤%疫苗%免疫效應
    갑태단백류%간종류%역묘%면역효응
    alpha-Fetoproteins%Liver neoplasms%Vaccines%Immunological effect
    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HLA-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4 h 51Cr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/105个细胞,均P ＜0.01],MAP的斑点数与阳性对照组[(166±32)斑点/105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/105个细胞,P＜0.01].结论 AFP MAP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.
    목적 탐토래원우인갑태단백( AFP)적세포독성T림파세포(CTL)표위다항원태(MAP)역묘대원발성간암적항종류활성.방법 체외분리배양인류백세포항원(HLA)-A2.1양성적건강지원자외주혈화HLA-A2.1전기인소서골수래원적성숙수돌세포(DC),용DC부재다태역묘,체내외유도효응세포,채용표준4 h 51Cr석방실험화매련면역반점기술(ELISPOT)검측CTL활성.결과 (1)재최고효파비시CTL표위MAP역묘유도적효응세포대AFP양성병차HLA-A2.1양성적Hep3B간암세포적살상솔현저고우단태표위유도적효응세포(73.5％±7.9％비45.6％±6.9％,P＜0.01).(2)최고효파비시AFP표위MAP역묘화단태역묘유도적효응세포대AFP음성이HLA-A2.1양성적PLC/PRF/5간암세포적살상솔여음성태대조조비교,차이균무통계학의의(9.3％±3.9％、8.1％±2.8％비8.3％±2.6％,균P＞0.05)；MAP조화대응단태조효응세포대전염AFP전장cDNA적PLC/PRF/5/Ad-AFP간암세포적살상솔균현저고우음성태대조조(74.8％±10.5％、51.4％±12.6％비4.2％±1.3％,균P＜0.01),이차MAP유도적CTL대PLC/PRF/5/AFP간암세포적살상솔현저고우상응단태(P＜0.01).(3)최고효파비시AFP표위MAP역묘화단태역묘유도적효응세포대AFP양성단HLA-A2.1음성적HepG2간암세포적살상솔여음성태대조조차이균무통계학의의(균P＞0.05)；기대전염료HLA-A2.1전장cDNA적HepG2/HLA-A2.1세포적살상솔칙균현저고우음성태대조조(71.8％±8.6％、46.5％±6.5％비4.1％±1.1％,균P＜0.01),MAP유도적CTL대HepG2/HLA-A2.1적살상솔현저고우상응단태(P＜0.01).(4)AFP표위MAP역묘화단태역묘유도적효응세포산생적IFN-γ반점수균현저고우음성대조태[(158 ±23)、(78±12)비(3±1)반점/105개세포,균P ＜0.01],MAP적반점수여양성대조조[(166±32)반점/105개세포]차이무통계학의의(P＞0.05),MAP적반점수역현저고우기단태[(78±12)반점/105개세포,P＜0.01].결론 AFP MAP유도적CTL반응강우상응단태유도적CTL반응,대AFP양성차HLA-A2.1상필배적종류세포구유명현적살상효응；대AFP음성적종류세포불구유살상효응.
    Objective To explore the antitumor effects of multiple antigen peptide (MAP) vaccine from α-fetoprotein (AFP) through AFP-specific cytotoxic T lymphocyte (CTL) against hepatoma in vitro and ex vivo.Methods Dendritic cells ( DC ) were generated from human perpheral blood mononuclear cells (PBMC) and HLA-A2.l-transgenic murine bone marrow.The AFP-specific CTL were induced by MAPloaded DC and the corresponding linear peptides from human AFP.The lysis rate of effectors to hepatoma cells were tested by 4h51Cr 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 epitopic MAP vaccines to Hep3B cells (AFP+,HLA-A2.1+ ) at the highest effector/target (E/T)ratio was significantly higher than linear peptide vaccine (73.5％ ±7.9％ vs 45.6％ ±6.9％,P ＜0.01 ).The effectors 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 cells transfected with cDNA 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 epitopic MAP vaccines was significantly higher than the corresponding linear peptide vaccine ( P ＜ 0.01 ). Compared with the negative control group,the effectors could not lyse HepG2 liver cancer cells,a HLA-A2.1 negative cell line ( both P ＞ 0.05 ).But the effectors could lyse HepG2 cells transfected with cDNA 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 effectors 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/105 cells ) or linear peptide vaccine group ((78 ± 12) spots/105 cells) were significantly higher than the negative control group ((3 ± 1)spots/105 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/105 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.
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