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

The CTL responce of anti-breast cancer induced by h MAM-A derived HLA-A2-restricted epitope peptide

目的:探讨h MAM-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^+/h MAM-A^+)AU-565、(HLA-A2^-/h MAM-A^+)MDA-MB-415、(HLA-A2^+/h MAM-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h MAM-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。

Objective： To investigate the cytotoxic activity of HLA-A2-restricted h MAM-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^＋/h MAM-A^＋） AU-565,（ HLA-A2^-/h MAM-A^＋） MDA-MB-415,（ HLA-A2^＋/h MAM-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： h MAM-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.