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目的以重组腺病毒载体携带免疫相关基因共刺激分子B7-1和粒细胞-巨噬细胞集落刺激因子(GM-CSF)转染神经母细胞瘤(NB)SH-SY5Y细胞株,观察其对肿瘤细胞免疫原性的影响。方法将携带绿色荧光蛋白的腺病毒质粒(Ad-GFP),以不同感染倍数(MOI)转染SH-SY5Y细胞,流式细胞仪检测腺病毒对该细胞株的转染效率。分别以空病毒质粒(Ad组)、携带B7-1和GM-CSF基因(B7-GM组)及仅携带B7-1基因(B7组)的腺病毒质粒转染SH-SY5Y细胞,并以未转染的肿瘤细胞为空白对照(SH组)。分别采用流式细胞仪(FCM)和ELISA检测B7-1分子和GM-CSF蛋白表达。Ficoll密度梯度离心法直接分离和纯化外周血单个核细胞,分别以效靶比51和101进行混合淋巴细胞培养后,测定放射性核素闪烁计数(cpm)/min,同时检测上清液中细胞因子IFN-γ及IL-2的分泌水平。结果腺病毒对SH-SY5Y细胞有较高的转染效率,转染后目的基因B7-1和GM-CSF得到了有效表达。B7-GM组、B7组cpm值及上清液IFN-γ、IL-2水平明显高于Ad组、SH组(Pa<0.01);B7-GM组与B7组比较,效靶比51与101比较,cpm值及细胞因子水平增高更明显(Pa<0.05)。结论B7-1和GM-CSF基因具有协同作用,可促进T淋巴细胞增殖,诱导细胞因子分泌水平增加,从而增强SH-SY5Y细胞的免疫原性。
OBJECTIVE: To investigate the effect of recombinant adenovirus vector carrying the costimulatory molecule B7-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on neuroblastoma (NB) SH-SY5Y cells. Effect of cellular immunogenicity. Methods The adenovirus vector carrying green fluorescent protein (Ad-GFP) was transfected into SH-SY5Y cells at different multiplicity of infection (MOI). The transfection efficiency of adenovirus on this cell line was detected by flow cytometry. SH-SY5Y cells were transfected with the empty virus plasmid (Ad group), the B7-1 and GM-CSF gene (B7-GM group) and the B7-1 gene (B7 group) Transfected tumor cells were blank control (SH group). Flow cytometry (FCM) and ELISA were used to detect the expression of B7-1 and GM-CSF. Peripheral blood mononuclear cells were directly isolated and purified by Ficoll density gradient centrifugation. After mixed lymphocyte culture with target ratios 51 and 101, respectively, the radionuclide scintigraphy (cpm) / min was measured. At the same time, the cytokines IFN-γ and IL-2 secretion levels. Results The adenovirus had high transfection efficiency on SH-SY5Y cells, and the target genes B7-1 and GM-CSF were efficiently expressed after transfection. B7-GM group, B7 group cpm value and the supernatant IFN-γ, IL-2 levels were significantly higher than the Ad group, SH group (Pa <0.01); B7-GM group compared with the B7 group, Comparison, cpm values and cytokine levels increased more significantly (Pa <0.05). Conclusion The B7-1 and GM-CSF genes have a synergistic effect, which can promote the proliferation of T lymphocytes and induce the increase of cytokine secretion, thereby enhancing the immunogenicity of SH-SY5Y cells.