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目的研究MAGE 3抗原肽对肝细胞癌 (HCC)患者进行免疫治疗的可行性。方法微量细胞毒法检测HCC患者中HLA A2表达情况 ,RT PCR方法检测肝癌组织MAGE 3基因mRNA表达情况。用免疫磁珠从患者外周血单个核细胞 (PBMC)中分离出CD8阳性细胞作为效应细胞 ;将其余自体CD8阴性的PBMC与MAGE 3抗原肽 (FLWGPRALV)短暂孵育 ,经γ射线照射后作为抗原呈递细胞 (APC) ,与CD8阳性效应细胞进行混合淋巴细胞培养 ,作为实验组 ,7d后再加入APC刺激患者效应细胞 1次。每周计数效应细胞数量 ,培养第 15天时作为细胞毒T淋巴细胞 (CTL)检测其杀伤活性。以表达MAGE 3和HLA A2的肝癌细胞系HLE作为靶细胞 ,采用IFN γ细胞因子分泌法 ,通过流式细胞仪检测具有分泌γ 干扰素 (IFN γ)的CD8阳性细胞的频数来反映CTL的杀伤活性。另设不加APC刺激的CD8阳性细胞 ,加IL 2培养作为对照组。结果本组 2 5例HCC患者中 9例HLA A2阳性表达 ;9例中 3例MAGE 3基因mRNA阳性表达 ,6例阴性表达。培养开始时 ,效应细胞为 1 0× 10 6/孔 ,2周后细胞增殖 4～ 6倍。分泌IFN γ的CD8阳性细胞的频数 ,3例MAGE 3阳性患者平均为 2 2 0 % ,6例阴性患者为 0 5 % ,两组间差异有非常显著意义 (P <0 0 1)。结论本实验结果表明 ,应用MAGE 3抗原肽 ,能在体外从HCC患者
Objective To study the feasibility of MAGE 3 antigen peptide immunotherapy in patients with hepatocellular carcinoma (HCC). Methods The expression of HLA A2 in HCC patients was detected by MTT assay. The mRNA expression of MAGE 3 gene in HCC tissues was detected by RT-PCR. CD8-positive cells were isolated as effector cells from peripheral blood mononuclear cells (PBMCs) of patients using immunomagnetic beads; the remaining autologous CD8-negative PBMCs were transiently incubated with MAGE 3 antigen peptide (FLWGPRALV) and irradiated as γ-rays for antigen presentation Cells (APCs) were cultured in mixed lymphocytes with CD8 positive cells. As experimental group, APCs were stimulated with stimulator cells once more after 7 days. The number of effector cells was counted weekly, and the cytotoxic T lymphocytes (CTLs) were tested for their killing activity on the 15th day of culture. Using HLE cells expressing MAGE 3 and HLA A2 as hepatocellular carcinoma cells, the frequency of CD8 positive cells secreting interferon gamma (IFNγ) was measured by flow cytometry to reflect CTL killing by IFN γ cytokine secretion active. Another set without additional APC stimulated CD8 positive cells, plus IL 2 culture as a control group. Results Of the 25 HCC patients, 9 were HLA A2 positive and 9 MAGE 3 mRNA was positive, while 6 were negative. At the beginning of culture, the effector cells were 10 × 10 6 / well, and the cells proliferated 4 to 6 times after 2 weeks. The frequency of CD8 positive cells secreting IFNγ was 220% in 3 cases of MAGE 3 positive patients and 0 5% in 6 negative patients. There was significant difference between the two groups (P <0.01). Conclusion The experimental results show that the application of MAGE 3 antigen peptide can be in vitro from HCC patients