树突状细胞疫苗特异肿瘤多肽联合树突状细胞体外刺激淋巴细胞功能评估

目的:利用树突状细胞(dendritic cell,DC)作为抗原递呈载体,检测树突状细胞疫苗特异肿瘤多肽在体外对淋巴细胞是否有刺激增殖、促进细胞因子分泌及促进对肿瘤细胞的杀伤功能。方法:外周血树突状细胞和细胞因子诱导的杀伤细胞(cytokine-induced killer,CIK)分离及培养采用贴壁法;采用CCK-8法检测淋巴细胞增殖功能;酶联免疫斑点法检测淋巴细胞的细胞因子分泌功能;CCK-8法检测淋巴细胞对肿瘤细胞的杀伤功能。实验分为肿瘤多肽组(肽+DC-CIK)、DC-CIK组和单纯CIK组进行各项功能检测与比较。结果:在白介素-2(interleukin-2,IL-2)存在时, 3组细胞均增殖明显。肿瘤多肽组在第4天、第6天(第4天Z=-3.79, P<0.001;第6天Z=-2.95, P<0.01)时,450 nm光密度显著高于CIK组,在第4天时,450 nm光密度显著高于DC-CIK组(Z=-2.02, P<0.05)。培养体系中无IL-2时,各组淋巴细胞均增殖缓慢,各时间点光密度差异无统计学意义。肿瘤多肽刺激组多种细胞因子产生量高于单纯CIK组,其中白介素-4(interleukin-4,IL-4)(Z=-2.61, P<0.01)、巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulation factor, GM-CSF)(Z=-3.85, P<0.001)、干扰素-γ(interferon-γ, IFN-γ)(Z=-3.56, P<0.001)和肿瘤坏死因子-α(tumor necrosis factor-α, TNF-ɑ, Z=-3.40, P<0.001)的分泌量与单纯CIK组相比,差异均有统计学意义。肿瘤多肽刺激组与DC-CIK组相比,除IL-4分泌量差异有统计学意义外(Z=-2.15, P<0.05), 其余各项细胞因子分泌量差异均无统计学意义。DC-CIK组与单纯CIK组相比,IFN-γ(Z=-2.44, P<0.05)、TNF-ɑ(Z=-2.26, P<0.05)和GM-CSF(Z=-3.73, P<0.001)分泌量差异有统计学意义。肿瘤多肽组与DC-CIK组、单纯CIK组在18 h与24 h的杀伤效率虽然差异无统计学意义,但有高于其他两组的趋势。结论:树突状细胞疫苗特异肿瘤多肽联合树突状细胞体外可提高CIK细胞的增殖活性,提高多个细胞因子的分泌量。

Assessment of lymphocytic function in vitro stimulated by specific tumor polypeptide combined with dendritic cells

Objective: To assess the activation function of specific tumor polypeptide for dendritic cell vaccine on lymphocytes proliferation, production of cytokines and killing activity in vitro by using dendritic cells as antigen presenting vector. Methods: Peripheral blood dendritic cells (DC) and cytokine-induced killer (CIK) were isolated and cultured by adherent culture method; CCK-8 method was used to assess the proliferation function of lymphocytes and the killing function of lymphocytes to tumor cells; enzyme-linked immunospot assay method was used to evaluate the secretion function of cytokines. The experiment was divided into tumor polypeptide group (peptide with DC-CIK), DC-CIK group and CIK group. Results: With presence of interleukin-2 (IL-2) in the culture system, the lymphocyte proliferation of the three groups was obvious. The absorbance at 450 nm of tumor polypeptide group was significantly higher than that of CIK group at the time points day 4 and day 6 (day 4: Z=-3.79, P<0.001; day 6: Z =-2.95, P< 0.01). The absorbance at 450 nm of group tumor polypeptide was significantly higher than that of DC-CIK group on day 4 (Z=-2.02, P<0.05). Without IL-2 in the culture system, lymphocytes proliferated slowly in all the three groups, and there was no significant difference in 450 nm absorbance at each time point. The levels of IL-4 (Z=-2.61, P< 0.01), granulocyte-macrophage colony-stimulation factor (GM-CSF, Z=-3.85, P< 0.001), interferon- γ (IFN- γ, Z=-3.56, P< 0.001) and tumor necrosis factor-α (TNF-ɑ, Z=-3.40, P< 0.001) of tumor polypeptide group were higher than those of CIK group. There was no significant difference in the production of cytokines except IL-4 (Z=-2.15, P< 0.05) when tumor polypeptide group was compared with DC-CIK group. The production of IFN-γ (Z=-2.44, P<0.05), TNF-ɑ (Z=-2.26, P< 0.05) and GM-CSF (Z=-3.73, P< 0.001) in DC-CIK group were higher than those of CIK group. Although there was no significant difference in killing activity between tumor polypeptide group, DC-CIK group and CIK group at hour 18 and hour 24, and the killing activity of tumor polypeptide group was higher than that of the other two groups. Conclusion: Tumor peptide combined with dendritic cells can improve the proliferation activity of CIK cells in vitro, and increase the secretion of several cytokines.