肿瘤细胞治疗研究
英文原题：MUC1 peptide-loaded dendritic cell vaccine boosts antitumor immunity in pancreatic cancer.
MUC1 肽脉冲的 DCs 有效激活特异性 CTL 反应，表明基于 DC 的疫苗免疫治疗在胰腺癌的管理中具有前景。
胰腺癌是侵袭性最强的恶性肿瘤之一，预后差且治疗选择有限。本研究旨在评估负载黏蛋白1（MUC1）肽抗原的树突状细胞（DC）疫苗在胰腺癌免疫治疗中的疗效。
从脐带血中分离单个核细胞，诱导分化为DC。采用流式细胞术检测DC表面标志物及其对FITC-OVA的吞噬能力。通过CCK-8法评估负载MUC1抗原肽（568和619）的DC疫苗对T淋巴细胞增殖的刺激作用。采用ELISA检测DC分泌IL-12p70及活化细胞毒性T淋巴细胞（CTLs）产生IFN-γ的情况。通过流式细胞术分析DC疫苗活化的CTLs中CD8 + 和CD4 + 细胞比例。采用LDH释放实验评估活化T细胞对胰腺癌细胞系的细胞毒活性。此外，进行生物信息学分析，比较胰腺癌与正常组织中MUC1表达及其与患者预后的相关性。采用Western blot检测胰腺癌细胞系中MUC1表达。在胰腺癌人源化huHSC-M-NSG小鼠模型中研究负载抗原肽619的DC疫苗的抗肿瘤效果。
未成熟DC（imDCs）高表达CD11c、HLA-DR和CD86，低表达CD14；成熟DC（mDCs）高表达CD11c、HLA-DR、CD83、CD80和CD86，低表达CD14。培养第5天的DC对FITC-OVA的吞噬能力最强。负载MUC1肽568或619的DC疫苗显著促进T淋巴细胞增殖，并诱导更高水平的IL-12p70和IFN-γ分泌。肽脉冲DC疫苗显著增加了CTLs中CD8+ T细胞的比例，并介导了对胰腺癌细胞系（PANC-1、BXPC-3、MIA PaCa-2）的剂量依赖性细胞毒性作用，其中MUC1肽619组效果最佳。生物信息学分析显示，MUC1在胰腺癌组织中高表达，并与患者不良预后相关。Western blot进一步证实了MUC1在胰腺癌细胞系中的表达。在体内，脉冲肽619的DC疫苗显著抑制了肿瘤生长（肿瘤重量抑制率：51.4%），增加了外周血中CD8+ T细胞的百分比，并增强了hCD45+细胞向肿瘤组织的浸润。
OBJECTIVE: Pancreatic cancer is one of the most aggressive malignancies with a poor prognosis and limited treatment options. This study aimed to evaluate the efficacy of a dendritic cell (DC) vaccine pulsed with mucin 1 (MUC1) peptide antigens in the immunotherapy of pancreatic cancer. METHODS: Mononuclear cells were isolated from umbilical cord blood and induced to differentiate into DCs. The surface markers of DCs and their phagocytic capacity for FITC-OVA were detected using flow cytometry. The stimulatory effect of DC vaccines loaded with MUC1 antigen peptides (568 and 619) on T lymphocyte proliferation was assessed by CCK-8 assay. ELISA was used to measure the secretion of IL-12p70 by DCs and IFN-γ production by activated cytotoxic T lymphocytes (CTLs). The proportion of CD8 + and CD4 + cells among CTLs activated by the DC vaccine was analyzed via flow cytometry. The cytotoxic activity of activated T cells against pancreatic cancer cell lines was evaluated using an LDH release assay. Furthermore, bioinformatic analysis was performed to compare MUC1 expression between pancreatic cancer and normal tissues and its correlation with patient prognosis. Western blot was used to detect MUC1 expression in pancreatic cancer cell lines. The antitumor effect of the DC vaccine pulsed with antigen peptide 619 was investigated in a humanized huHSC-M-NSG mouse model of pancreatic cancer. RESULTS: Immature DCs (imDCs) highly expressed CD11c, HLA-DR, and CD86, but weakly expressed CD14; mature DCs (mDCs) highly expressed CD11c, HLA-DR, CD83, CD80, and CD86, and weakly expressed CD14. DCs on day 5 of culture exhibited the strongest phagocytic capacity for FITC-OVA. DC vaccines loaded with either MUC1 peptide 568 or 619 significantly promoted T lymphocyte proliferation and induced higher levels of IL-12p70 and IFN-γ secretion. The peptide-pulsed DC vaccines significantly increased the proportion of CD8 + T cells among CTLs and mediated dose-dependent cytotoxic effects against pancreatic cancer cell lines (PANC-1, BXPC-3, MIA PaCa-2), with the highest efficacy observed in the MUC1 peptide 619 group. Bioinformatic analysis revealed that MUC1 was highly expressed in pancreatic cancer tissues and associated with poor patient prognosis. Western blot further confirmed MUC1 expression in pancreatic cancer cell lines. In vivo , the DC vaccine pulsed with peptide 619 significantly suppressed tumor growth (tumor weight inhibition rate: 51.4%), increased the percentage of CD8 + T cells in peripheral blood, and enhanced the infiltration of hCD45 + cells into tumor tissues. CONCLUSION: MUC1 peptide-pulsed DCs effectively activate specific CTL responses, indicating that DC-based vaccine immunotherapy holds promise for the management of pancreatic cancer.
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