B7-1和B7-2基因转染神经母细胞瘤细胞免疫原性的研究

Research of B7-1 and B7-2 genes cotransfection on immunogenicity of neuroblastoma cell lines

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 observe the effect of cotransduction of B7-1 and B7-2 genes mediated by recombinant adenovirus on cell immunogenicity of NB cell line SH-SY5Y.Methods： We transfected SH-SY5Y cells with recombinant adenovirus which carrying GFP（Ad-GFP）,and estimate the transfection efficiency of adenovirus by using flow cytometry（FCM）,then figure out the proper multiplicity of infection（MOI）,recombinant adenovirus mediating Ad、human B7-1 gene or B7-2 gene.Ad group and untransfected neuroblastoma cells was used as the control group.We investigated the protein expression of B7-1 and B7-2 by FCM.Isolation and purification PBMC by using Ficoll density gradient centrifu.The mix-cultivation of lymphocytes were cultured as the anti-target ratio of 5 ∶ 1 or 10 ∶ 1,then we tested the scintillation spectrometry of radionuclides（cpm/min）.Meanwhile,we measure the secretion level of IFN-γ and IL-2 in supernate.Results： After transfection with B7-1 and B7-2 genes were expressed,the level of IFN-γ、IL-2 and cpm was higher than that of Ad and SH group（P0.01）.No significant difference about the level of IFN-γ、IL-2 and cpm was observed between the B7-1 and B7-2 group（P0.05）.The level of IFN-γ、IL-2 and cpm was increase obviously when the anti-target ratio is 5 ∶ 1 or 10 ∶ 1（P0.05）.Conclusions： After transfection with B7-1 or B7-2,the proliferation and activation of T cells were apparently increased,and they can secrete more IFN-γ and IL-2 than the control group.But the level of IFN-γ、IL-2 and cpm have no significant difference between the B7-1 and B7-2 group.