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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 the specific anti-tumor effect of vaccination with dexamethasone 1 (MUC1) sensitized dendritic cells (DCs) using CpG as adjuvant. Methods Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation in healthy volunteers. DCs were induced by cytokines such as GM-CSF, IL-4 and TNF-α in vitro. The morphology of cells was observed by flow cytometry The mature DC cell markers CD80 and CD86 were detected. Then, Cp G was used as an adjuvant, MUC1 was used as antigen to sensitize DC to prepare vaccine, and DCs were mixed with T cells to observe their ability of inducing T lymphocyte proliferation. The cytotoxic T lymphocytes (CTLs) induced by CTLs were incubated with tumor target cells at target ratios of 5: 1, 10: 1 and 20: 1. MTT assay was used to detect CTL killing activity. Results The results of DC phenotype showed that CD80 + cells accounted for 70.4% and CD86 + cells accounted for 72.0%, showing a mature DC phenotype. MUC1-sensitized DC vaccine mixed with lymphocytes showed that DC vaccine could stimulate the proliferation and activation of T cells. The DC + Cp G + MUC1 group was significantly higher than MUC1 + DC or Cp G + DC group, the difference was statistically significant (P <0.01). The killing effect of CTL induced by DC vaccine on tumor cells was significantly higher than that of T cells or DC alone (P <0.01). And with the target ratio increased, the killing effect was gradually increased. Conclusions DC vaccines primed with Cp G and MUC1 induce specific anti-tumor immune responses in vitro.