【摘要】 目的:探讨肿瘤睾丸抗原NY-ESO-1(New York-esophageal-1)致敏树突状细胞体外诱导特异性CTL对肝癌细胞株的杀伤作用。方法:重组质粒pGEX-ESO1经原核诱导表达并纯化GST-ESO1融合蛋白肽。重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素4(rhIL-4)诱导培养人外周血来源的树突状细胞(dendritic cells,DCs),经GST-ESO1融合蛋白肽致敏后诱导特异性CTL增殖。以此CTL为效应细胞,分别以NY-ESO-1阳性表达的肝癌细胞株HepG2和不表达NY-ESO-1的肝癌细胞株H2P为靶细胞,MTT法检测CTL对肝癌细胞株的杀伤作用。结果:重组质粒pGEX-ESO1经IPTG诱导,在大肠杆菌中表达相对分子质量约36 000的GST-ESO1融合蛋白肽,纯化后的质量浓度为50μg/ml;经rhGM-CSF和rhIL-4联合诱导成功培养人外周血DCs,其表型分子HLA-DR为91.4%、CD86为70.5%、CD83为71.2%、CD80为55.3%。NY-ESO-1致敏的DCs能明显诱导CTL增殖,此CTL对肝癌细胞株HepG2的杀伤率显著高于GST刺激组、未致敏DC组和无DC刺激组(均P<0.05),效靶比为50∶1时杀伤效应达到最高峰[(53.23±3.78)%,P<0.01];相同条件下CTL对H2P细胞无特异性杀伤作用。结论:NY-ESO-1抗原致敏的DCs在体外可诱导同种CTL产生和增殖,后者对NY-ESO-1阳性肝癌细胞株具有特异性杀伤效应,该方法为肝癌免疫治疗提供了一条新思路。更多还原

【Abstract】 Objective:To study the antigen specific anti-tumor effect of cytotoxic T lymphocyte(CTL),which was induced by cancer testis antigen NY-ESO-1-impulsed dendritic cells(DCs),against human hepatocellular carcinoma(HCC).Methods: GST-ESO1 fusion protein was induced in recombinant pGEX-ESO1 vector transformed bacteria by IPTG,and the GST-ESO1 fusion protein was purified.DCs were induced with granulocyte/macrophage colony-stimulating factor(GM-CSF) and interleukin-4(IL-4) from human peripheral blood mononuclear cells.DCs impulsed with GST-ESO protein peptide were co-cultured with T lymphocytes,and the resultant CTLs were used as effector cells.NY-ESO-1 positive hepatocellular carcinoma HepG2 cells and NY-ESO-1 negative H2P cells were used as target cells to test the specific anti-tumor effect of CTL using MTT.Results: Escherichia coli BL21 expressed fusion protein peptide GST-ESO1(Mr 36 000) after transfection with recombinant pGEX-ESO1 vector.The concentration of GST-ESO1 peptide was 50 μg/ml after purification.DCs were successfully induced with GM-CSF and IL-4 from human peripheral blood mononuclear cells.DCs-impulsed with NY-ESO1 had high expression of surface molecule such as HLA-DR(91.4%),CD86(70.5%),CD83(71.2%) and CD80(55.3%).DCs-impulsed with NY-ESO-1 induced production and proliferation of CTL,and this CTL specifically killed NY-ESO-1 positive HepG2 cells.CTL induced by NY-ESO-1 had stronger cytotoxic effect against HepG2 cells compared with GST-impulsed DCs,un-impulsed DCs(P<0.05).Highest anti-tumor activity was found when the ratio of effector∶target was 50 ∶1(53.23±3.78,P<0.01).Conclusion: DCs-impulsed with NY-ESO-1 can induce production and proliferation of allogenic CTLs,which show antigen specific anti-tumor effect against NY-ESO-1 positive HCC cells.This result casts new lights on immunotherapy of HCC.更多还原