hMAM-A源性HLA-A2限制表位肽诱导CTL对乳腺癌细胞的杀伤作用

摘要

Objective:To investigate the cytotoxic activity of HLA-A2-restricted hMAM-A derived candidate epitope peptide(KLLMVLMLAA) pulsed dendritic cells(DCs) induced the cytotoxic lymophocyte(CTL) on breast cancer.Methods:HLA-A2+ healthy volunteers peripheral blood was obtained,using density gradient centrifugation to separate peripheral blood mononuclear cell(PBMC).In vitro,DCs were divided to 2 groups:peptide-pulsed DCs group,no peptide-pulsed DCs group.DCs surface markers were determined by flow cytometry.IL-10,IL-12,IFN-γ secretion levels were determined by ELISA.Cytotoxic activity of CTL on(HLA-A2+/hMAM-A+)AU-565,(HLA-A2-/hMAM-A+)MDA-MB-415,(HLA-A2+/hMAM-A-)MCF-7 was determined by CCK8.Results:Mature DCs were successfully cultivated with typical morphological and functionality characteristics.The expression of CD80,CD86,CD83,HLA-DR induced peptide-pulsed DCs were little higher than the control group.At the same time,the average of IL-12 was higher,the average of IL-10 was lower,IFN-γ secretion levels were significantly higher than the control group(P<0.05).Cytotoxic activity of peptide-pulsed DCs induced CTL on AU-565 was significantly better than the control group(MCF-7,MDA-MB-415).Conclusion:hMAM-A derived HLA-A2-restricted CTL epitope peptide can successful was predicted and effectively rendered by DC and CTL,can produce specific lysis on breast cancer cell.%目的:探讨hMAM-A源性HLA-A2限制性候选表位(KLLMVLMLAA)负载外周血树突状细胞(dendritic cells,DCs)诱导产生的细胞毒性杀伤细胞(CTL)对乳腺癌细胞的杀伤作用.方法:取HLA-A2+健康志愿者外周血,用密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC),体外用合成的肽段负载DC,不加肽的DC作为对照,采用流式细胞术测定DC的表型变化,ELISA测定IL-10、IL-12、IFN-γ分泌水平.CCK8法测定两种情况下DC诱导的自体CTL对(HLA-A2+/hMAM-A+)AU-565、(HLA-A2-/hMAM-A+)MDA-MB-415、(HLA-A2+/hMAM-A-)MCF-7乳腺癌细胞的杀伤效果.结果:成功培养出形态典型、功能成熟的DC,经肽负载的DC具有更典型的形态特征,DC表面标记分子(CD80、CD86、CD83、HLA-DR)较对照组稍高,上清中IL-12平均分泌水平较对照组高,IL-10平均分泌水平较对照组低,与T细胞共培养后IFN-γ分泌水平较对照组显著增高(P<0.05),对AU-565的杀伤效果显著优于MDA-MB-415、MCF-7乳腺癌细胞.结论:成功预测出hMAM-A源性HLA-A2+限制性CTL表位肽,其致敏的DC可有效呈递表位肽,诱导的CTL对乳腺癌细胞产生特异性杀伤.