黏蛋白1(MUC1)致敏的树突状细胞疫苗体外抗肿瘤实验研究

Experimental Study on the Antitumor Effects in Vitro of Dendritic Cell Vaccine Pulsed with Mucin1(MUC1)

目的研究以Cp G作为免疫佐剂,应用黏蛋白1(MUC1)致敏树突状细胞(DC)制备疫苗在体外诱导的特异性抗肿瘤作用。方法健康人外周血采用密度梯度离心法,分离外周血单核细胞(PBMC),应用GM-CSF、IL-4、TNF-α等细胞因子体外培养诱导DC,观察细胞形态,并通过流式细胞术检测成熟DC细胞标志物CD80、CD86。再应用Cp G作为免疫佐剂,MUC1作为抗原致敏DC制备疫苗,致敏的DC与T细胞混合培养,观察其诱导T淋巴细胞增殖能力。诱导产生的细胞毒性T淋巴细胞(CTL)与肿瘤靶细胞以5∶1、10∶1、20∶1的效靶比共孵育,MTT法检测CTL杀伤活性。结果 DC表型检测结果为CD80+细胞占70.4%,CD86+细胞占72.0%,呈现成熟DC表型。MUC1致敏的DC疫苗与淋巴细胞混合培养显示,DC疫苗具有刺激T细胞增殖活化的作用,其中DC+Cp G+MUC1组明显高于MUC1+DC或Cp G+DC组,差异有统计学意义(P<0.01)。DC疫苗诱导产生的特异性CTL对肿瘤细胞的杀伤作用较单独T细胞组或DC组明显增强,差异有统计学意义(P<0.01)。并随着效靶比增大,其杀伤作用呈逐渐增强。结论经Cp G和MUC1致敏的DC疫苗在体外可诱导产生特异性抗肿瘤免疫效应。

Objective To study CpG as immune adjuvant,application of Mucinl - sensitization dendritic cells（DC） vaccine to in- duce specific antitumor effect in vitro. Methods DCs were isolated from peripheral blood mononuclear ceils （PBMCs） in vitro. The CD80 and CD86 markers of mature DCs were detected by flow cytometry. CpG was used as immune adjuvant,MUC1 was used as antigen to induce DC vaccine, and DC vaccine were mixed with T cells to induce T lymphocyte proliferation. The cytotoxic T lymphocytes （CTLs） were incubated with tumor target cells at the ratios of 5： 1,10： 1,20： 1. MTT assay was used to detect the cytotoxlcity of CTL. Results CDS0^＋ cells accounted for 70.4% and CD86 ^＋ cells accounted for 72.0% of the DC phenotype, showing a mature DC phenotype. DC vaccine and lymphocyte mixed culture showed that the DC vaccine had the effect of stimulating T cell proliferation and activation. DC ＋ CpG ＋ MUC1 group was significantly higher than MUC1 ＋ DC or CpG ＋ DC group（P 〈 O. 01 ）. The specific CTL induced by DC vaccine on tumor cells was significantly higher than that in DC alone or in T cell group （ P 〈 0.01 ）. And with the effeetor - target ratio increased, the killing effect was gradually increased. Conclusion MUC1 sensitized DC vaccine can induce specific antitumor immune effect in vitro, combined with CpG can further enhance the antitumor effect of DC vaccine.