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50 μg/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。

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.

福建省自然科学基金资助项目（No. C0610023）；广州市科技计划资助项目(No. 2003J1C0221)