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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 cytotoxicity of cytotoxic killer (CTL) induced by dendritic cells (DCs) loaded with hMAM-A-derived HLA-A2-restricted candidate epitope (KLLMVLMLAA) on breast cancer cells . Methods: The peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers with HLA-A2 + and dendritic cells (DCs) were cultured in vitro. The phenotypic changes of DCs were detected by flow cytometry. The levels of IL-10, IL-12 and IFN-γ were measured by ELISA. CCK8 assay was used to detect the effect of DC-induced autologous CTL on the expression of HLA-A2 + / h MAM-A + AU-565 and HLA-A2- / h MAM- A + MDA- , (HLA-A2 ~ + / h MAM-A ~ -) MCF-7 breast cancer cells. RESULTS: DCs with typical morphological and functional characteristics were successfully cultivated. The DCs with peptide-loaded DCs had more typical morphological characteristics. The surface markers (CD80, CD86, CD83, HLA-DR) -12, the average secretion level of IL-10 was lower than that of the control group, and the level of IFN-γ secretion was significantly higher than that of the control group (P <0.05). The killing effect of AU-565 Significantly better than MDA-MB-415, MCF-7 breast cancer cells. CONCLUSION: The hMAM-A-derived HLA-A2 ~ + restricted CTL epitope peptide was successfully predicted. The sensitized DCs could effectively deliver the epitope peptide, and the induced CTLs could specifically kill breast cancer cells.