目的探讨应用MAGE-1抗原肽治疗肝细胞肝癌（HCC）的可行性。方法 应用RT-PCR法检测MAGE-1在8种HCC细胞株中的表达，筛选杀伤实验的靶细胞；将MAGE-1抗原肽NYKCRFPEI孵育的外周血单个核白细胞经照射后作为抗原呈递细胞，每隔7天刺激HCC病人自体外周血单个核白细胞一次，共4次后作为效应T淋巴细胞，应用流式细胞仪检测培养前后淋巴细胞的表型变化，应用乳酸脱氢酶法检测效应T细胞对靶细胞的杀伤效应。 结果 HCC细胞株BEL7405 MAGE-1和HLA-A24均阳性表达，可用作杀伤实验的阳性靶细胞，HLE等其它7种细胞不能双表达，可用作杀伤实验的阴性对照细胞；培养28天，淋巴细胞增加31倍；培养28天，CD3+ T和CD8+ T分别增加26%和20%；在效∶靶为10∶1时，效应T细胞对MAGE-1抗原九肽NYKCRFPEI孵育的自体淋巴母细胞、BEL7405的杀伤效应分别为62.5%和40.25%，均明显高于对自体淋巴母细胞（17.88%）、HLE（19.55%）及QGY7701的杀伤效应（1.6%）；在效∶靶为3.3∶1时，效应T细胞对肽孵育的自体淋巴母细胞的杀伤效应为53.6%，明显高于对自体淋巴母细胞（15.6%）、HLE（13%）和QGY7701的杀伤效应（1%）。 结论 本实验结果表明，应用MAGE-1抗原肽NYKCRFPEI，在体外从HCC病人的外周血单个核白细胞中成功地诱导出具有特异性杀伤能力的效应T细胞, 为应用MAGE-1抗原肽对HCC病人进行免疫治疗提供了理论基础。%Objective To investigate the possibility of using melanoma antigen-1 (MAGE-1) peptide as a tumor vaccine to treat hepatocellular carcinoma (HCC). Methods The expressions of MAGE-1 in 8 HCC cell lines and in liver cancer tissue from a patient were detected using RT-PCR. The type of human leucocyte antigen Ⅰ (HLAⅠ) of both 8 HCC cell lines and peripheral blood mononuclear cells of the patient was detected using a microcytotoxicity method to screen out target cell lines for the cytotoxicity assay. Peripheral blood mononuclear cells from the HCC patient pulsed with an MAGE-1 peptide (NYKCRFPEI) were used as antigen presenting cells. Autogenous peripheral blood mononuclear cells were stimulated with antigen presenting cells every 7 days for 4 times to elicit cytotoxic T lymphocytes. The phenotype of effector cells was analyzed using flow cytometry. The cytotoxicity of effector cells was detected with a lactate dehydrogenase releasing assay.Results The expressions of both MAGE-1 and HLA-A24 were detected in BEL7405 cell line which were used as the positive target cell line in the cytotoxicity assay. The expression of MAGE-1 alone was detected in HLE, BEL7402, BEL7404, QGY7703 and SMMC7721 cell lines, and the expression of neither MAGE-1 nor HLA-A24 was shown in QGY 7701 and HpG2 cell lines. The last 7 cell lines could be used as negative target cell lines in the cytotoxicity assay. Peripheral blood mononuclear cells expanded 32 folds during 28-day culture. The ratio of CD3+ T cells increased by 16% (from 54% to 70%), and the ratio of CD8+ T cells increased by 20% (from 36% to 56%) during 28-day culture. When the ratio of effector cells to target cells was 10∶1, effector cells exhibited 62.5% cytotoxicity against autogenous lymphoblasts pulsed with the peptide (NYKCRFPEI) of MAGE-1 antigen, 40.25% cytotoxicity against BEL7405 cells, compared with 17.88% cytolysis observed against autogenous lymphoblasts, 19.55% against HLE cells, and 1.6% against QGY7701 cells. When the ratio of effector cells to target cells was 3.3∶1, the cytotoxicity of effector cells against the peptide pulsed autogenous lymphoblasts was 53.6%, which was much higher against autogenous lymphoblasts, HLE cells and QGY7701 cells at 15.6%, 13% and 1%, respectively.Conclusion The results demonstrate that cytotoxic T lymphocytes with the ability to specifically lyse target cells expressing both MAGE-1 and HLA-A24 could be successfully induced by the MAGE-1 peptide NYKCRFPEI in vitro. This indicates that a good result might be anticipated if this peptide is used as a tumor vaccine to treat HLA-A24 HCC patients.
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