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目的比较免疫共刺激信号B7-1和B7-2以腺病毒为载体转染神经母细胞瘤(NB)SH-SY5Y细胞株,观察其对肿瘤细胞免疫原性的影响。方法以携带绿色荧光蛋白的腺病毒质粒(Ad-GFP)转染SH—SYSY细胞,通过流式细胞仪(FCM)判断转染效率,确定合适的感染倍数(MOI)。分别以空病毒质粒(Ad)及携带B7-1或B7-2的腺病毒质粒转染SH-SYSY细胞,即对照组和B7-1或B7-2组;并以未转染的肿瘤细胞为空白对照(空白组)。采用流式细胞仪(FCM)检测B7-1分子和B7-2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5∶1和10∶1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B7-2得到了有效表达。B7-1组、B7-2组cpm值及上清液IFN-γ、IL-2水平明显高于Ad组、SH组(P<0.01);B7-1组与B7-2组比较,cpm值及细胞因子水平增高无明显差别(P>0.05)。效靶比5∶1时,与10∶1相比,cpm值及IFN-γ、IL-2含量的增加更明显(P<0.05)。结论 B7-1和B7-2分子可促进T淋巴细胞的增殖及活化T细胞分泌IFN-γ、IL-2,而B7-1与B7-2两组间cpm值及细胞因子水平无明显差别。
Objective To compare the immunostimulatory signals B7-1 and B7-2 with adenovirus vector transfected neuroblastoma (NB) SH-SY5Y cell lines to observe its impact on tumor cell immunogenicity. Methods SH-SYSY cells were transfected with adenovirus vector carrying green fluorescent protein (Ad-GFP), and the transfection efficiency was determined by flow cytometry (FCM) to determine the appropriate multiplicity of infection (MOI). SH-SYSY cells were transfected with empty virus plasmid (Ad) and adenovirus vector carrying B7-1 or B7-2 respectively, that is, control group and B7-1 or B7-2 group. The untransfected tumor cells were Blank control (blank group). The expression of B7-1 and B7-2 proteins were detected by flow cytometry (FCM). Peripheral blood mononuclear cells were directly isolated and purified by Ficoll density gradient centrifugation. Mixed lymphatic After the cell culture, the radionuclide scintigraphy (cpm) / min was measured, and the secretion levels of the cytokines IFN-γ and IL-2 in the supernatant were measured. Results The adenovirus had high transfection efficiency on SH-SY5Y cells, and the target genes B7-1 and B7-2 were efficiently expressed after transfection. The cpm values of B7-1 group and B7-2 group and the levels of IFN-γ and IL-2 in supernatant were significantly higher than those in Ad group and SH group (P <0.01). Compared with B7-1 group, cpm value And cytokines levels increased no significant difference (P> 0.05). Compared with 10: 1, the cpm value and the increase of IFN-|Ã and IL-2 levels were more obvious (P <0.05). Conclusion B7-1 and B7-2 molecules can promote the proliferation of T lymphocytes and the secretion of IFN-γ and IL-2 by activated T cells. There is no significant difference in cpm and cytokine between B7-1 and B7-2.